Fourier and power law analysis of structural complexity in cornea and lens

The ordered pattern of type I collagen fibrils in the transparent cornea is an example of specialization in the formation of functional ultrastructure. In contrast, the disordered and amorphous distribution of cytoplasmic proteins in the transparent lens resembles the structure of most cells. While the organization of cytoplasmic proteins is often considered to be random, the compartmentalization of functional proteins in biological cells and the organization provided by cytoskeletal elements suggests that non-random patterns of organization are common. Attempts to quantify disordered, amorphous patterns of ultrastructure in cells and tissues have been unsuccessful, in part, because the cellular organization of structural proteins including collagen, keratin, cytoskeletal and crystallin proteins is complex. Characterization of the complex patterns observed in electron micrographs is a fundamental problem in structural biology. This paper reviews the use of Fourier and power law analyses of electron micrographs of cornea and lens as models for ordered and disordered ultrastructure of cells and tissues.     

Contributions to osteoclast biology from Japan

Bone is a dynamic tissue, in which bone formation by osteoblasts and bone resorption by osteoclasts continue throughout life. In 1998, we molecularly cloned osteoclast differentiation factor (ODF), a long-thought factor responsible for osteoclast formation. This review article describes how Japanese scientists contributed to osteoclast biology before and after the discovery of ODF. This review article is based on the Louis V. Avioli Memorial Lecture of the American Society for Bone and Mineral Research (ASBMR) held in Honolulu in September, 2007.     

Strategies and results of field emission scanning electron microscopy (FE-SEM) in the study of parasitic protozoa

Field emission scanning electron microscopy (FE-SEM) provides a range of strategies for investigating the structural organization of biological systems, varying from isolated macromolecules to tissue organization and whole organisms. This review covers some of the results so far obtained using FE-SEM observation and various protocols of sample fixation to analyze the structural organization of parasitic protozoa and their interaction with host cells. The employment of FE-SEM can be broadened through the use of gold-labeled molecules or tracers, gradual extraction by detergents, and cleavage techniques. These analyses provide significant contributions to the characterization of these organisms concerning ultrastructure, cytoskeleton, motility and intracellular behavior.     

Interaction of endothelial cells with biodegradable strontium-doped calcium polyphosphate for bone tissue engineering

Angiogenesis is of great importance in bone tissue engineering, and has gained large attention in the recent 10 years. However, little research has been done on the effect of biodegradable materials, especially their degradation products on the angiogenesis process. Strontium-doped calcium polyphosphate (SCPP) has been proved to be able to promote osteoblasts growth in vitro before. In the present work, the interaction of endothelial cells (ECs) with the scaffold of SCPP was investigated to evaluate its potential influences on angiogenesis. The cell adhesion on SCPP scaffold as well as the angiogenic behaviors including proliferation, migration and tube-like structure (TLS) formation of ECs treated by its degradation products was tested. The results were compared with those of CPP group and physiological saline (negative control). As the results showed, the surface of SCPP could promote the adhesion and spreading of ECs. Ca²⁺ and Pi as well as Sr²⁺ were the main degradation products of SCPP. They did not inhibit but could promote the proliferation of ECs within 90 days. Moreover, they could induce the migration and TLS formation of ECs. Since SCPP bears the ability to improve the adhesion and angiogenic behaviors of endothelial cells, it might benefit angiogenesis and serve as a more promising scaffold for bone tissue engineering application. Besides, this work may provide a new method for in vitro evaluation of biodegradable materials’ potential effects on angiogenesis.     

Measurement of Strain Distributions in Mouse Femora with 3D-Digital Speckle Pattern Interferometry

Bone is a mechanosensitive tissue that adapts its mass, architecture and mechanical properties to external loading. Appropriate mechanical loads offer an effective means to stimulate bone remodeling and prevent bone loss. A role of in situ strain in bone is considered essential in enhancement of bone formation, and establishing a quantitative relationship between 3D strain distributions and a rate of local bone formation is important. Digital speckle pattern interferometry (DSPI) can achieve whole-field, non-contacting measurements of microscopic deformation for high-resolution determination of 3D strain distributions. However, the current system does not allow us to derive accurate strain distributions because of complex surface contours inherent to biological samples. Through development of a custom-made piezoelectric loading device as well as a new DSPI-based force calibration system, we built an advanced DSPI system and integrated local contour information to deformation data. Using a mouse femur in response to a knee loading modality as a model system, we determined 3D strain distributions and discussed effectiveness and limitations of the described system.     

Synchrotron diffraction study of deformation mechanisms in mineralized tendon

The high stiffness and toughness of biomineralized tissues are related to the material deformation mechanisms at different levels of organization, from trabeculae and osteons at the micrometer level to the mineralized collagen fibrils at the nanometer length scale. Quantitatively little is known about the sub-micrometer deformation mechanisms under applied load. Using a parallel-fibred mineralized tissue from the turkey leg tendon as a model for the mineralized collagen fibrils, we used in situ tensile testing with synchrotron x-ray diffraction to measure the average fibril deformation with applied external strain. Diffraction peak splitting occurred at large strains, implying an inhomogeneous elongation of collagen fibrils. Scanning electron microscopy measurements lead us to conclude that the inhomogeneous mineralization in mineralized tendon is at the origin of the high fracture strain.     

Ultrastructure of spermatogenesis in the white-lined broad-nosed bat, Platyrrhinus lineatus (Chiroptera: Phyllostomidae)

Spermatogenesis, the remarkable process of morphological and biochemical transformation and cell division of diploid stem cells into haploid elongated spermatozoa, is one of the most complex cell differentiations found in animals. This differentiation process has attracted extensive studies, not only because the process involves many radical changes in the cell shape and biochemistry, but also because the phases and steps of differentiation have provided a better basis for analyzing the seminiferous epithelium cycle. Thus, this study aimed to characterize ultrastructurally the spermatogenesis process in the bat Platyrrhinus lineatus in order to provide a basis for determining the stages of spermatogenesis and to facilitate comparisons of the process between bat species and other vertebrates. Based on ultrastructural characteristics three main types of spermatogonia could be accurately identified: A(d), A(p) and B; the differentiation of spermatids was clearly divided into 12 steps (steps 1-3: Golgi phase, steps 4-5: cap phase, steps 6-9: acrosomal phase and steps 10-12: maturation phase). The ultrastructure of spermatozoa, Leydig cells and Sertoli cells was characterized; and some processes including nucleolar disorganization and the formation of synaptonemal complexes, acrosome and chromatoid body were discussed. Based on our results we may conclude that the spermatogenic process of P. lineatus follows the pattern of mammals with some specificity, as the process of formation of the acrosome and the presence of the perfuratorium. By other side, the simpler ultrastructure of its spermatozoon shows a pattern more closely related to the sperm cells of humans and other primates.     

Bone microarchitecture evaluated by histomorphometry

The increasing use of densitometric devices for assessing bone fragility has progressively strengthened the assumption that mass is the most important property determining bone mechanical competence. Nevertheless, structure and microarchitecture are relevant aspects of bone strength. The study of microarchitecture is based on the measure of width, number, and separation of trabeculae as well as on their spatial organization. There are several methods to assess bone architecture, particularly at the trabecular level. In particular, histomorphometry, based on the use of optical microscopy and on the principles of quantitative histology and stereology, evaluates microarchitecture two-dimensionally, even if these measures appear well correlated to the three-dimensional structure and properties of bone. In addition, new computerized methods allow the acquisition of more sophisticated measurements by means of a digitizer have been introduced to integrate the use of the microscope. These methods supply information on trabecular width as well as on its distribution and on the organization of the trabeculae in the marrow space.

Microarchitecture seems to be a determinant of bone fragility independent of bone density and it is important for understanding the mechanisms of bone fragility as well as the action of the drugs used to prevent osteoporotic fractures. Several in vivo studies (on animals and humans) can provide an additional interpretation for the anti-fracture effect of such drugs. For instance, bisphosphonates and parathyroid hormone seem to preserve or even improve microarchitecture. The challenge for the future will be to evaluate bone quality in vivo with the same or better resolution and accuracy than the invasive methods used today.     

A short flagella mutant of Dunaliella sallina (Chlorophyta, Cholorophyceae)

Dunaliella salina (Chlorophyta, Chlorophyceae) is a unicellular wall-less biflagellate alga. In this paper we describe a spontaneous mutant of D. salina, isolated from wild type cultures, which is characterized by very short flagella. The ultrastructure showed the basic 9 + 2 organization of wild-type flagella. Immunofluorescence localization of tubulin in this mutant confirmed the normal construction of the axoneme. Although, the mutant does not swim, still it is able to move and perform photobehavior. As shown by track reconstruction, and rotation movements, observed by means of reflection microscopy, this mutant can move, probably gliding by means of its stumpy flagella. A possible model to explain the mutant motion pattern is discussed.     

A spatiotemporal master equation model of morphogen transport: Local accumulation times,noise measurement and diffusion force

Morphogen, a class of signaling molecules to direct and control pattern formation of cell and tissue, is first synthesized in a local region and then conveyed to other regions or degraded. In the previous studies, this transport process was modeled by deterministic models of ordinary differential equations. In microcosmic environments, however, the process is often affected by stochastic fluctuations (or the noise). It remains unclear how this noise affects morphogen gradients. Here, we build a spatiotemporal master equation model for the process of morphogen transport in a finite developmental field, from which we derive the first-order moment equations of this master equation. We derive the analytical expression of the local accumulation time that the morphogens reach a steady state, and find that this time is nonlinear with respect to the cell positions. We also derive the approximate expressions of the steady-state variances, the Fano factors and the local accumulation time of the variance. Interestingly, we find that the local accumulation time for the variance of the morphogen number is shorter than that of its corresponding second-order moment. Moreover, the noise in the morphogen number is almost not affected by the distance from the cellular position to morphogen source. In addition, we further study some quantities (e.g., potential energy and diffusion force) from the view of physical-chemical mechanisms, and uncover that the diffusion force is a key factor for the formation of the morphogen gradient. Our results provide insights on morphogen diffusion.     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

A method for vibrational assessment of cortical bone

Large bones from many anatomical locations of the human skeleton consist of an outer shaft (cortex) surrounding a highly porous internal region (trabecular bone) whose structure is reminiscent of a disordered cubic network. Age related degradation of cortical and trabecular bone takes different forms. Trabecular bone weakens primarily by loss of connectivity of the porous network, and recent studies have shown that vibrational response can be used to obtain reliable estimates for loss of its strength. In contrast, cortical bone degrades via the accumulation of long fractures and changes in the level of mineralization of the bone tissue. In this paper, we model cortical bone by an initially solid specimen with uniform density to which long fractures are introduced; we find that, as in the case of trabecular bone, vibrational assessment provides more reliable estimates of residual strength in cortical bone than is possible using measurements of density or porosity.     

Vertebral cancellous bone turn-over: microcallus and bridges in backscatter electron microscopy

Backscatter electron microscopy (BSE) is a powerful technique for investigating cancellous bone structure. Its main function is to offer information regarding the degree of mineralization of the tissue within individual trabeculae.

To illustrate the qualitative information that can be drawn from BSE imaging technique, we present a study on human vertebral cancellous bone. This tissue is continuously remodeled through osteoclastic resorption and osteoblastic new bone apposition. It is thought that osteoclastic resorption pits are especially deleterious for vertebral bone architecture since they often perforate the thin trabeculae; the osteoblasts being unable to repair the gap. In addition, excessive stress may also disrupt the architecture in case of trabecular fracture or damage accumulation.

Waves of new bone formation were easy to identify in BSE. Often these waves were connecting both edges of a perforation and called bridges. Additionally, we present a few images of microcallus formations. A microcallus is described as a small mass of woven bone that generally repairs a trabecula. The microstructural aspects of different microcalluses are presented and discussed. Both bridges and microcallus should be considered as examples of the repair porcess since they obviously preserve the connectivity of the trabeculae. However, bridges were much more frequent than microcallus (396 vs 15). Both mechanisms probably illustrate the normal response to different local stimuli.     

Atomic Force Microscopy of bulk tendon samples: affect of location and fixation on tissue ultrastructure

Atomic Force Microscopy (AFM) is a surface characterisation technique which analyses topology. To date, AFM studies of tissue ultrastructure have focussed on single collagen fibrils extracted from different tissues prior to analysis. Using sample preparation techniques used in electron microscopy studies, this work uses AFM to analyse the collagen ultrastructure of bulk samples from bovine deep digital flexor tendons (DDFTs). DDFT ultrastructure in regions of the tendon which experience different loading conditions are compared. Samples are analysed post-freezing and post-aldehyde fixation with either 10% formalin or 4% glutaraldehyde in order to investigate the affect of tissue preservation on tissue ultrastructure. The results demonstrate that both fibril diameter and repeat unit of the tendon vary between different regions in the dorsoventral plane, with regions subjected to both tensile and compressive forces exhibiting smaller fibril diameter and repeat unit compared to regions subjected to tensile forces alone. These differences are detectable regardless of the tissue preservation technique used. However these measured differences do vary with preservation techniques with aldehyde-fixed samples exhibiting smaller fibril diameters and larger repeat units compared to frozen samples. These results demonstrate that AFM is a highly suitable technique for the characterisation of different ultrastructures in bulk samples but that it is important to be consistent in the choice of preservation technique.     

Changes in ultrastructure and histochemistry of two red macroalgae strains of Kappaphycus alvarezii (Rhodophyta, Gigartinales), as a consequence of ultraviolet B radiation exposure

Ultraviolet radiation (UVR) affects macroalgae in many important ways, including reduced growth rate, reduction of primary productivity and changes in cell biology and ultrastructure. Among red macroalgae, Kappaphycus alvarezii is of economic interest by its production of kappa carrageenan. Only a few reports have examined the changes in macroalgae ultrastructure and cell biology resulting from UVB radiation exposure. Therefore, we examined two strains of K. alvarezii (green and red) exposed to UVB for 3 h per day during 28 days and then processed them for histochemical and electron microscopy analysis. Reaction with Toluidine Blue showed an increase in the thickness of the cell wall and Periodic Acid-Schiff stain showed a decrease in the number of starch grains. UVBR also caused changes in the ultrastructure of cortical and subcortical cells, which included increased thickness of the cell wall and number of free ribosomes and plastoglobuli, reduced intracellular spaces, changes in the cell contour, and destruction of chloroplast internal organization. Based on these lines of evidence, it was evident by the ultrastructural changes observed that UVBR negatively affects intertidal macroalgae and, by extension, their economic viability.     

Microstructure,crystallography and diagenetic alteration in fossil ostrich eggshells from Upper Palaeolithic sites of Indian peninsular region

Biominerals studies are of importance as they provide an understanding of natural evolutionary processes. In this study we have investigated the fossil ostrich eggshells using Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD) and Electron Backscatter Diffraction (EBSD). SEM studies demonstrated the ultrastructure of fossil eggshells and formation of calcified cuticular layer. The presence of calcified cuticle layer in eggshell is the basis for ancient DNA studies as it contains preserved biomolecules.EBSD accentuates the crystallographic structure of the ostrich eggshells with sub-micrometer resolution. It is a non-destructive tool for evaluating the extent of diagenesis in a biomineral. EBSD analysis revealed the presence of dolomite in the eggshells. This research resulted in the complete recognition of the structure of ostrich eggshells as well as the nature and extent of diagenesis in these eggshells which is vital for genetic and paleoenvironmental studies.     

Histomorphometric analysis of glucocorticoid-induced osteoporosis

Bone histomorphometry or quantitative histology consists of counting or measuring tissue components: cells, extracellular constituents and microarchitecture. Bone histomorphometry is the only method that allows the measurement of mineralization rate and the study of bone formation at three levels: cell, remodeling unit and tissue levels. It is a useful tool to explain the pathogenesis and cellular mechanisms of different metabolic bone diseases such as glucocorticoid-induced osteoporosis (GIO).

Glucocorticoids (GC) affect calcium and bone metabolism at every level, but the main effect is the osteoblastic dysfunction.

Concerning the bone formation, some histomorphometric studies have shown a depressed osteoblastic activity at a cell, bone remodeling unit, and tissue levels. In addition, there is evidence of a shortening of the period in which the osteoblasts work actively forming the bone matrix. This latter effect seems to occur after high cumulative doses of GC. With regard to the resorption, the results are still debated, but histomorphometric parameters seem to be increased in the majority of studies, at least in the first period of the GC treatment. From a structural point of view, GC seem to induce a thinning of the trabeculae without their perforation, which occurs only after high cumulative doses. Antiresorptive treatments, such as bisphosphonates, are able to counteract the negative effects of GC on bone. In particular, along with their active working period, they prolong the lifespan of osteoblasts and osteocytes. In addition, the antiresorptive treatments seem to extend the time for secondary mineralization through a reduction of the Activation Frequency. The latter is an intriguing mechanism of bisphosphonates in GIO that needs further ad hoc investigations.     

Nature-inspired surface topography: design and function

Learning from nature has traditionally and continuously provided important insights to drive a paradigm shift in technology.In particular,recent studies show that many biological organisms exhibit spectacular surface topography such as shape,size,spatial organization,periodicity,interconnectivity,and hierarchy to endow them with the capability to adapt dynamically and responsively to a wide range of environments.More excitingly,in a broader perspective,these normally neglected topological features have the potential to fundamentally change the way of how engineering surface works,such as how fluid flows,how heat is transported,and how energy is generated,saved,and converted,to name a few.Thus,the design of nature-inspired surface topography for unique functions will spur new thinking and provide paradigm shift in the development of the new engineering surfaces.In this review,we first present a brief introduction to some insights extracted from nature.Then,we highlight recent progress in designing new surface topographies and demonstrate their applications in emerging areas including thermal-fluid transport,anti-icing,water harvesting,power generation,adhesive control,and soft robotics.Finally,we offer our perspectives on this emerging field,with the aim to stimulate new thinking on the development of next-generation of new materials and devices,and dramatically extend the boundaries of traditional engineering.     

XRF analysis of strontium: Exploring cellulose as a soft tissue equivalent

X-ray fluorescence (XRF) is a widely used method for in vivo elemental analysis. Particularly for bone, it is a non-invasive technique that provides information on composition without significant risk to the patient. XRF contributes a capability for measuring elements beneficial to human health, such as strontium. This is a proposed supplement that has been shown in clinical trials to reduce fracture risk in people diagnosed with osteoporosis. Although XRF is a viable method for quantifying bone strontium, there are still factors that constrain its effectiveness. X-ray attenuation through overlying soft tissue decreases the signal, consequently requiring correction before estimating the true concentration of strontium in bone. A correction factor can be applied to account for the reduced signal, but an accurate measurement of overlying soft tissue thickness is required. It has been shown that using the correlation between Compton peak count rate and overlying thickness can be used as an estimation of overlying tissue. Lucite is commonly used as a soft tissue substitute; however, its mean atomic number is appreciably lower than soft tissue, somewhat limiting its applicability. This study tests the feasibility of using cellulose filter papers as a substitute for overlying soft tissue to perform XRF analysis of strontium-doped hydroxyapatite bone phantoms. Mass attenuation coefficients are shown to be closer to those of soft tissue (International Commission on Radiation Units' four-component) than Lucite, and the Compton correlation is used to estimate thickness as a correction factor to quantify true strontium concentration.     

Strength and Fracture Toughness of Polyurethane Elastomers Modified with Carbon Nanotubes

Very small additions of single-wall carbon nanotubes produce an anomalous change in the mechanical properties of a cross-linked polyurethane-amide-urea elastomer containing 10% of polyamide-6: its elastic modulus and ultimate stress reveal local maxima at a nanofiller content of hundredths and thousandths of a percent. Previously, the behavior of the elastic modulus was simulated reasoning from the formation of an intermediate phase layer in the elastomer at particle contact boundaries. Here, on the same basis, we simulate the behavior of its strength as a function of nanotube concentration and consider crack models accounting for the influence of nanotubes on the crack tip zone and fracture toughness.