High-resolution imaging of proteins in human teeth by scanning probe microscopy

High-resolution studies of dental tissues are of considerable interest for biomedical engineering and clinical applications. In this paper, we demonstrate the application of piezoresponse force microscopy (PFM) to nanoscale imaging of internal structure of human teeth by monitoring the local mechanical response to an electrical bias applied via a conductive tip. It is shown that PFM is capable of detecting dissimilar components of dental tissues, namely, proteins and calcified matrix, which have resembling morphology but different piezoelectric properties. It is demonstrated that collagen fibrils revealed in chemically treated intertubular dentin exhibit high piezoelectric activity and can be visualized in PFM with spatial resolution of 10 nm. Evidence of the presence of protein inclusions of 100-200 nm wide and several micrometers long in tooth enamel has been obtained. Furthermore, it is found that the peritubular dentin and intertubular dentin exhibit different piezoelectric behavior suggesting different concentration of collagen fibrils. The obtained results demonstrate a high potential of PFM in providing an additional insight into the structure of dental tissues. It is suggested that the PFM approach can be used to study the structure of a wide range of biological materials by monitoring their electromechanical behavior at the nanoscale.     

Structure and formation of ankylosis in Xenopus laevis

The structure of ankylotic teeth in Xenopus laevis was studied by light, transmission, and scanning electron microscopy as well as by microradiography in decalcified and undecalcified specimens. The mature teeth of Xenopus laevis are calcified from the crown to the base, fused to the jaw bone, and have no uncalcified area, such as a fibrous ring separating the tooth into the crown and pedicle. Microradiography shows that the mature tooth and jaw bone appear as an X-ray opaque area, except for the basal region of the dentine. This region is composed of an X-ray translucent area and an X-ray opaque thin layer on the lingual side of the translucent area. The mature tooth is composed of two differently calcified areas: (1) a highly calcified area, which makes up almost all of the tooth and contains a thin layer of the basal dentine on the lingual side, and (2) a lowly calcified basal dentine, which is fused to the jaw bone. Therefore, the lowly calcified area does not completely separate the dentine and jaw bone. Repeating banding patterns among the collagen fibrils differ among the dentine-forming area and the matrices of dentine and jaw bone. During the formation of ankylosis of the tooth germ, collagen bundles in the dentine-forming area accumulate directly on the surface of the jaw bone. Consequently, the mature teeth of Xenopus laevis fuse to the jaw bone directly without the mediation of the other structures.     

Structure of comblike teeth of the ayu sweetfish,Plecoglossus altivelis (Teleostei: Isospondyli): I. Denticles and tooth attachment

The structure and tooth attachment of the comblike teeth and denticles of the ayu sweetfish, Plecoglossus altivelis, were examined by light and scanning electron microscopy. The denticle is composed of a spoonlike crown with a spine pointed anteriorly, a triangular plate in the cervical region, and a root that curves laterally and tapers off to a point. The root apex is fused with a long thin pedicle that turns abruptly anteriad toward the jaw bone. Planes of the spine, the spoonlike crown, the triangle plate and the root of the denticle are varied, and the denticle is twisted in the region of the triangle plane. The superficial layer of the dentine is homogeneously calcified and is considered to be enameloid, because some of the inner dentinal epithelial cells in the tooth germ are columnar and possess cellular processes at their apical ends. The dentine is fibrous and fine dentinal tubules are visible in dentine treated with sodium hydroxide and observed by scanning electron microscopy. The upper half of the root is surrounded by a dense layer of collagen fibers running parallel to the tooth axis, and the lower half is encompassed by interlaced collagen fibers. The lower part of the root is open on its lingual side. The pedicle is a long rod which is homogeneously calcified and enmeshed by interlaced collagen fibers, and it curves mediad as it nears the jaw bone. The pedicles are interposed between a layer of gelatinous connective tissue and the jaw bone and terminate on the periosteum. Comparative aspects of ayu tooth morphology are discussed. © 1993 Wiley-Liss, Inc.     

A morphological study of regeneration of the goldfish elasmoid scales

Studies were carried out on the model of regeneration of mechanically removed elasmoid scales. Regenerating scales were morphologically analyzed using light and electron microscopy. It was found that the cells responsible for regeneration of the elasmoid scale plates could be classified as osteogenic elements. Little differentiated preosteoblasts were detected in the connective tissue of dermis on day 3 of regeneration, while partially calcified plates underlaid osteoblasts on day 7. The scale cover was fully restored on day 14 and it took two days for each bone plate to be formed. Osteocytes, fully differentiated osteogenic elements, were found in the deepest regions of newly formed scales.     

A morphological study of regeneration of the goldfish elasmoid scales

Studies were carried out on the model of regeneration of mechanically removed elasmoid scales. Regenerating scales were morphologically analyzed using light and electron microscopy. It was found that the cells responsible for regeneration of the elasmoid scale plates could be classified as osteogenic elements. Little differentiated preosteoblasts were detected in the connective tissue of dermis on day 3 of regeneration, while partially calcified plates underlay osteoblasts on day 7. The scale cover was fully restored on day 14 and it took two days for each bone plate to be formed. Osteocytes, fully differentiated osteogenic elements, were found in the deepest regions of newly formed scales.     

Facile preparation of biodegradable chitosan derivative having poly(butylene glycol adipate) side chains

Various modes are being explored for the construction of functional materials from nanoparticles. Despite these efforts, the assembly of nanoparticles remains challenging with respect to the requirement of multiple component organization on varying dimensions and length scales. The graft copolymers of chitosan with poly(butylene glycol adipate) (PBGA) were prepared due to the esterification reaction between PBGA and 6-O-succinate-N-phthaloyl-chitosan (PHCSSA) in the presence of toluene as a swelling agent. The graft copolymers are nanoparticles with the size of few hundred nanometers as observed from TEM. It is a potential method to combine chitosan with the hydrophobic synthetic polymers. The grafting reactions were conducted with various PBGA/PHCSSA feed ratios to obtain chitosan-g-PBGA copolymers with various PBGA contents. FT-IR, NMR, XRD, spectrofluorophotometer, and TEM were detected to characterize the copolymers.     

Mesoscale Modelling

Abstract

Increasingly, industrial materials are being designed to have structure on length scales of tens to thousands of nanometers. These structures are crucial to achieving a particular desired material property. Such structures, however, may depend on the underlying chemistry of the material for their existence. For example, a thousandfold increase in the ionic conductivity of a polymer blend may only occur in a narrow region of a hugely complex phase diagram, the location' of which region can be expected to depend on the molecular chemistry and physics from the monomer scale to the coil size.

Traditional Computational Chemistry has proved incapable of dealing with the length and time scales involved in the formation of these ‘Mesoscale’ structures. On the other hand, traditional Computational Physics has proved incapable of consistently incorporating the necessary chemical detail for modelling real industrial materials. In this paper we present two novel methods which successfully address both the chemistry and the physics of mesophase formation. The methods, described in detail, are MesoDyn and Dissipative Particle Dynamics (DPD).

Unlike phenomenological theories of materials, such as the Landau models which one finds in much of the computational physics literature, the two models mentioned incorporate molecular geometry and connectivity explicitly. We discuss each of the methods briefly.

We then give an overview of how these methods are being used in industry to optimise materials and processes. We discuss previous simulation results for triblock Pluronic surfactants in solution studied with MesoDyn, and for diblock copolymers studied with DPD, where the known experimental changes in morphology from micellar to hexagonal to bicontinuous to lamellar have been successfully reproduced. We also present new results for several systems, including binary and ternary blends, where the third component in the latter system is a diblock copolymer, which acts as a compatibiliser. We discuss the effects of changing solvent character on the material properties of these systems, as well as the effects of an externally imposed shear flow.     

Visualizing nature at work from the nano to the macro scale

Visalizing the structure and dynamics of proteins, supramolecular assemblies, and cellular components are often key to our understanding of biological function. Here, we focus on the major approaches in imaging, analyzing, and processing biomedical data ranging from the atomic to the macro scale. Relevant biomedical applications at different length scales are chosen to illustrate and discuss the various aspects of data acquisition using multiple modalities including electron microscopy and scanning force microscopy. Moreover, powerful scientific software is presented for processing, analyzing, and visualizing heterogeneous data. Examples of using this software in the context of visualizing biological nano-machines are presented and discussed.     

Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components

The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.     

First Shark from the Late Devonian (Frasnian) Gogo Formation,Western Australia Sheds New Light on the Development of Tessellated Calcified Cartilage

BackgroundLiving gnathostomes (jawed vertebrates) comprise two divisions, Chondrichthyes (cartilaginous fishes, including euchondrichthyans with prismatic calcified cartilage, and extinct stem chondrichthyans) and Osteichthyes (bony fishes including tetrapods). Most of the early chondrichthyan (‘shark’) record is based upon isolated teeth, spines, and scales, with the oldest articulated sharks that exhibit major diagnostic characters of the group—prismatic calcified cartilage and pelvic claspers in males—being from the latest Devonian, c. 360 Mya. This paucity of information about early chondrichthyan anatomy is mainly due to their lack of endoskeletal bone and consequent low preservation potential.Conclusions/SignificanceThe Meckel’s cartilages show a jaw articulation surface dominated by an expansive cotylus, and a small mandibular knob, an unusual condition for chondrichthyans. The scapulocoracoid of the new specimen shows evidence of two pectoral fin basal articulation facets, differing from the standard condition for early gnathostomes which have either one or three articulations. The tooth structure is intermediate between the ‘primitive’ ctenacanthiform and symmoriiform condition, and more derived forms with a euselachian-type base. Of special interest is the highly distinctive type of calcified cartilage forming the endoskeleton, comprising multiple layers of nonprismatic subpolygonal tesserae separated by a cellular matrix, interpreted as a transitional step toward the tessellated prismatic calcified cartilage that is recognized as the main diagnostic character of the chondrichthyans.     

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth-anchoring structure. Periodontal ligament is a unique non-mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)-positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over-expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast-related cells, but it also regulates cementoblast commitment in periodontal ligament cells.     

Transformation of the viscoelastic functions of calcified tissues and interfacial biomaterials into a common representation

Since both connective and calcified tissues are markedly viscoelastic in nature, an understanding of the behavior of these tissues intrinsically as materials on their own, as well as in composite formation with synthetic implants, is of prime importance in order to predict and anticipate materials' design and function. Thus considerable interest has developed in recent years with respect to measurements of the viscoelastic properties of biological materials. However, attempts to characterize the viscoelasticity of calcified tissues have involved many different experimental procedures; hence results appear in terms of different functions, e.g. relaxation modulus, creep compliance. Since this diversity precludes a simple useful comparison of the results, the present study was initiated so that measured functions could be cast into a common representation, and thus compared. Linear viscoelasticity theory implies definite exact relationships between the functions. Using these relations, experimental results on bone, dentin and implant materials presently used to interface to the natural tissues, e.g. polymethyl methacrylate and high density linear polyethylene, were transformed into the complex dynamic modulus representation. Analysis shows that the results of experiments on bone are not in agreement as to dispersion (i.e. change of modulus with frequency) and its variation with strain. Further, analysis of the internal consistency of some experiments demonstrates a violation of the Boltzmann integral which indicates that linear viscoelasticity (almost invariably assumed by workers in the field) fails for bone in compression. It is concluded that the dynamic behavior of bone is not as well understood as has been thought heretofore; direction is given for future experiments. Contribution No. 59 from the Laboratory for Crystallographic Biophysics; supported by USPHS through NIDR Grant Number 5T1-DE-117-10.     

Transient and microscale deformations and strains measured under exogenous loading by noninvasive magnetic resonance

Characterization of spatiotemporal deformation dynamics and material properties requires non-destructive methods to visualize mechanics of materials and biological tissues. Displacement-encoded magnetic resonance imaging (MRI) has emerged as a noninvasive and non-destructive technique used to quantify deformation and strains. However, the techniques are not yet applicable to a broad range of materials and load-bearing tissues. In this paper, we visualize transient and internal material deformation through the novel synchrony of external mechanical loading with rapid displacement-encoded MRI. We achieved deformation measurements in silicone gel materials with a spatial resolution of 100 μm and a temporal resolution (of 2.25 ms), set by the repetition time (TR) of the rapid MRI acquisition. Displacement and strain precisions after smoothing were 11 μm and 0.1%, respectively, approaching cellular length scales. Short (1/2 TR) echo times enabled visualization of in situ deformation in a human tibiofemoral joint, inclusive of multiple variable T(2) biomaterials. Moreover, the MRI acquisitions achieved a fivefold improvement in imaging time over previous technology, setting the stage for mechanical imaging in vivo. Our results provide a general approach for noninvasive and non-destructive measurement, at high spatial and temporal resolution, of the dynamic mechanical response of a broad range of load-bearing materials and biological tissues.     

Morphology of the tooth of the American alligator (Alligator mississippiensis): The fine structure and elemental analysis of the cementum

The fine structure and the concentration of trace elements in the cementum layer in functional teeth of subadult alligators (ca. 120 cm to 160 cm total length) was studied by using scanning electron microscopy (SEM), microradiography, and electron microprobe analysis (EMPA). The cementum layer was hypertrophic and consisted of two layers: the fibrous layer and the calcified layer. The two layers undergo developmental changes as a result of resorption and replacement. During the tooth replacement in the American alligator, trace elements decreased at the base of the dentine layer; the resorption of the alveolar bone occurred simultaneously at the tooth socket. We concluded that the resorption of the cementum in the alligator provided a useful indication of the mechanism of tooth replacement in crocodilian.     

The functions of mechanosensitive ion channels in tooth and bone tissues

Tooth and bone are independent tissues with a close relationship. Both are composed of a highly calcified outer structure and soft inner tissue, and both are constantly under mechanical stress. In particular, the alveolar bone and tooth constitute an occlusion system and suffer from masticatory and occlusal force. Thus, mechanotransduction is a key process in many developmental, physiological and pathological processes in tooth and bone. Mechanosensitive ion channels such as Piezo1 and Piezo2 are important participants in mechanotransduction, but their functions in tooth and bone are poorly understood. This review summarizes our current understanding of mechanosensitive ion channels and their roles in tooth and bone tissues. Research in these areas may shed new light on the regulation of tooth and bone tissues and potential treatments for diseases affecting these tissues.     

Effect of polyelectrolyte structure on protein-polyelectrolyte coacervates: coacervates of bovine serum albumin with poly(diallyldimethylammonium chloride) versus chitosan

Electrostatic interactions between synthetic polyelectrolytes and proteins can lead to the formation of dense, macroion-rich liquid phases, with equilibrium microheterogeneities on length scales up to hundreds of nanometers. The effects of pH and ionic strength on the rheological and optical properties of these coacervates indicate microstructures sensitive to protein-polyelectrolyte interactions. We report here on the properties of coacervates obtained for bovine serum albumin (BSA) with the biopolyelectrolyte chitosan and find remarkable differences relative to coacervates obtained for BSA with poly(diallyldimethylammonium chloride) (PDADMAC). Coacervation with chitosan occurs more readily than with PDADMAC. Viscosities of coacervates obtained with chitosan are more than an order of magnitude larger and, unlike those with PDADMAC, show temperature and shear rate dependence. For the coacervates with chitosan, a fast relaxation time in dynamic light scattering, attributable to relatively unrestricted protein diffusion in both systems, is diminished in intensity by a factor of 3-4, and the consequent dominance by slow modes is accompanied by a more heterogeneous array of slow apparent diffusivities. In place of a small-angle neutron scattering Guinier region in the vicinity of 0.004 A-1, a 10-fold increase in scattering intensity is observed at lower q. Taken together, these results confirm the presence of dense domains on length scales of hundreds of nanometers to micrometers, which in coacervates prepared with chitosan are less solidlike, more interconnected, and occupy a larger volume fraction. The differences in properties are thus correlated with differences in mesophase structure.     

Assessment of 90Sr concentration in dental tissue using thin-layer beta-particle detectors and verification with numerical calculations

Electron paramagnetic resonance (EPR) measurements of tooth enamel can be used as an individual biological dosimeter for external dose assessment. However, the presence of 90Sr in the tooth tissues makes the task of interpreting EPR tooth dosimetry more complicated. The determination of the dose contribution of incorporated 90Sr in calcified tissue to the total dose measured by EPR is one of the main aspects of correct interpretation of EPR tooth dosimetry. In this work, experimental and numerical calculations were performed to convert the measured beta-particle dose rate to 90Sr concentration in calcified tissue. The cumulative beta-particle dose was measured by exposing artificially contaminated dentin and enamel to thin-layer alpha-Al2O3:C detectors in two different exposure geometries. Numerical calculations were performed for experimental exposure conditions using calculations of electron transport and secondary photons [Monte Carlo n-Particle Transport code version 4C2 (MCNP)]. Numerical calculations were performed to optimize the sample size and exposure geometry. The applicability of two different exposure conditions to be used in routine analysis was tested. Comparison of the computational and experimental results demonstrated very good agreement.     

Label‐free multi‐parametric imaging of single cells: dual picosecond optoacoustic microscopy

Advances in microscopy with new visualization possibilities often bring dramatic progress to our understanding of the intriguing cellular machinery. Picosecond optoacoustic micro‐spectroscopy is an optical technique based on ultrafast pump‐probe generation and detection of hypersound on time durations of picoseconds and length scales of nanometers. It is experiencing a renaissance as a versatile imaging tool for cell biology research after a plethora of applications in solid‐state physics. In this emerging context, this work reports on a dual‐probe architecture to carry out real‐time parallel detection of the hypersound propagation inside a cell that is cultured on a metallic substrate, and of the hypersound reflection at the metal/cell adhesion interface. Using this optoacoustic modality, several biophysical properties of the cell can be measured in a noncontact and label‐free manner. Its abilities are demonstrated with the multiple imaging of a mitotic macrophage‐like cell in a single run experiment.