Osteoclast diseases

Osteoclasts are the only cells capable of resorbing mineralised bone, dentine and cartilage. Osteoclasts act in close concert with bone forming osteoblasts to model the skeleton during embryogenesis and to remodel it during later life. A number of inherited human conditions are known that are primarily caused by a defect in osteoclasts. Most of these are rare monogenic disorders, but others, such as the more common Paget's disease, are complex diseases, where genetic and environmental factors combine to result in the abnormal osteoclast phenotype. Where the genetic defect gives rise to ineffective osteoclasts, such as in osteopetrosis and pycnodysostosis, the result is the presence of too much bone. However, the phenotype in many osteoclast diseases is a combination of osteosclerosis with osteolytic lesions. In such conditions, the primary defect is hyperactivity of osteoclasts, compensated by a secondary increase in osteoblast activity. Rapid progress has been made in recent years in the identification of the causative genes and in the understanding of the biological role of the proteins encoded. This review discusses the known osteoclast diseases with particular emphasis on the genetic causes and the resulting osteoclast phenotype. These human diseases highlight the critical importance of specific proteins or signalling pathways in osteoclasts.     

Unique dentition of lungfish

Lungfish are a group of ancient fish, represented almost continuously in the fossil record from their first appearance in the Devonian to the present time. They have numerous unique characters in the dentition, found only among lungfish, as well as some that are shared with other groups of fish and with higher vertebrates, such as a thin layer of true enamel, instead of the enameloid found in the dentition of most fish. Unique characters include the diversity of forms of dentine in the tooth plates, as well as the organisation of the dentines and the unusual mode of continuous growth of the dentition. Their enamel is based on amelogenin formed by the ameloblasts of the dental lamina, and is mineralised by long crystals of calcium hydroxyapatite deposited in layers over the developing tooth. The different forms of dentine, which show progressive evolutionary change within the group, are based on an extracellular matrix of collagen, mineralised with calcium hydroxapatite of variable crystalline form. Cells that line the oral cavity produce the ameloblasts that are endodermal in origin, and cells that form the dental papillae are, like the other cells that secrete skeletal tissues, derived from head mesoderm, as in other vertebrates. Unique to all dipnoans is the fusion of the dental tissues to the underlying bone and the way in which bone, included within the tooth plate, remodels to permit growth in the whole dentition.     

Quantitative analyses of the biomineralization of different hard tissues

The primary crystallites of the different developing hard tissues have an apatite structure. However, they have crystal lattice distortions representing an intermediate state between amorphous and fully crystalline. We have applied energy-filtering transmission electron microscopy in the selected area electron diffraction mode to analyse different stages of crystal formation in dentine, bone, enamel and inorganic apatite mineral. We have obtained quantitative information on the degree of crystal lattice distortion using the paracrystal theory of Hosemann and Bagchi.
We have found that the early formed crystallites of the hard tissues being analysed have a paracrystalline character comparable to biopolymers. However, with maturation, the lattice fluctuations of the crystallites of the hard tissues bone, enamel and dentine decrease to form a typical (para)crystalline character. Also the decrease of the organic proportion in the matrix corresponds to the decrease of the lattice fluctuation of the crystallites in the different hard tissues during maturation.     

High-resolution surface scanning electron microscopy of mineralized tissues

By using an ion-pumped SEM with high brightness gun, resolution of the order of 5 nm has been demonstrated on the mineral matrix of animal bone and tooth enamel. Measurements of collagen fibril diameter have also been obtained.     

Scanning and transmission electron microscopy for evaluation of order/disorder in bone structure

A comparative characterization of the structure of normal and abnormal (osteoporotic) human lumbar and thoracic vertebrae samples was carried out to reveal the type of possible disorder. Samples from the bone fragments extracted during the surgery due to vertebra fractures were examined by scanning electron microscopy (SEM), conventional and high resolution transmission electron microscopy (TEM and HRTEM), and X-ray energy dispersive spectroscopy (EDS). Contrary to what might be expected in accordance with possible processes of dissolution, formation and remineralization of hard tissues, no changes in phase composition of mineral part, crystal sizes (length, width, and thickness), and arrangement of crystals on collagen fibers were detected in abnormal bones compared to the normal ones. The following sizes were determined by HRTEM for all bone samples: 相似文献   

Image analysis of mineralized and non-mineralized type I collagen fibrils

Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen-associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm axial repeat of the gap/overlap zone plus four other maxima corresponding to repeat spacings of 22, 16, 13, and 11 nm respectively. Computer imaging techniques were used to reconstruct images by using selected spatial frequencies from such transforms. In this manner the subperiodic distributions of mineral were visually enhanced. These subperiodicities are positioned in an asymmetric fashion over the entire D unit repeat aligning with the molecular orientation of the fibril. Analyses of both negatively stained collagen and computer-generated maps of collagen hydrophobicity were compared to the mineral distribution of collagen. Densitometric comparisons showed a positional correlation between the axial banding patterns of mineralized fibrils and those of negatively stained non-mineralized fibrils. Comparable spatial frequencies were also present in transforms between hydrophobic maps and mineral distribution of collagen. These results suggest that the lateral clusterings of hydrophobic residues which span the fibril at specific sites in both the gap and overlap zones serve to prohibit early mineral deposition. This observed hydrophobic influence in combination with the gap space appear as contributing factors in the observed axial distribution of mineral within collagen.     

Calcium silicate cement‐induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate‐based restorative material (Biodentine^TM) and a glass ionomer cement (GIC:?Fuji?IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate‐free tetracycline, and tetracycline‐free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8‐week storage at 37 °C, dentine samples were imaged using two‐photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine^TM in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra‐ and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement‐free or PBS‐free media, or with GIC. It could therefore be concluded that Biodentine^TM induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate‐rich media, which was selectively detectable using the tetracycline labelling.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Multiscale characterization of pathological bone tissue

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.     

A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC-5) cells

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.     

Physico-chemical characterization of functional electrospun scaffolds for bone and cartilage tissue engineering

Mimicking the zonal organization of the bone-cartilage interface will aid the production of functional osteochondral grafts for regeneration of skeletal joint defects. This study investigates the potential of the electrospinning technique to build a three-dimensional construct recapitulating the zonal matrix of this interface. Poly(lactic-co-glycolic acid) (PLGA) and PLGA-collagen solutions containing different concentrations of hydroxyapatite nanoparticles (nHAp) were electrospun on a thin layer of phosphate buffer saline solution spread on the collector in order to facilitate membrane detachment and recovery. Incorporation of increasing amounts of nHAp in PLGA solutions did not affect significantly the average diameter of the fibres, which was about 700 nm. However, in the presence of collagen, fibres with diameters below 100 nm were generally observed and the number of these fibres was inversely proportional to the ratio PLGA:collagen and proportional to the content of nHAp. PLGA membranes were rather hydrophobic, although the aqueous drop contact angles progressively fell from 125 degrees to 110 degrees when the content of nHAp was increased from 0 per cent to 50 per cent (w/v). PLGA-collagen membranes were more hydrophilic with contact angles between 60 degrees and 110 degrees; the values being proportional to the ratio PLGA:collagen and the content of nHAp. Also, the addition of nHAp from 0 per cent to 50 per cent (w/v) in the absence of collagen resulted in decreasing dramatically both the Young's modulus (Ym), from 34.3 +/- 1.8 MPa to 0.10 +/- 0.06 MPa, and the ultimate tensile strain (epsilon max), from a value higher than 40 per cent to 5 per cent. However, the presence of collagen together with nHAp allowed the creation of membranes much stiffer, although more brittle, as shown for membranes made with a ratio 8:2 and 10 per cent of nHAp, for which Ym = 70.0 +/- 6.6 MPa and epsilon max = 7 per cent.     

The application of EDXS to the biological sciences

The distribution of chemical elements in soft tissues may be faithfully preserved by very rapid freezing. Most often the material is then cryosectioned and the sections frozen-dried prior to analysis, but direct analysis in the hydrated state is an established alternative. For bulk specimens, the shape of the analysed volume is uncertain. But whichever current model is accepted, analytical spatial resolution must generally be limited to the order of 1 μm. Such specimens can be suitable for the specific analysis of cytoplasm, cell nuclei and large extracellular spaces but not for study on a finer scale. Analytical spatial resolution in the range 200–500 nm is obtainable with sections cut ～ 1 μm thick. In the frozen-hydrated state, small extracellular spaces can be analysed but multiple scattering obscures intracellular detail in the STEM image. The irradiation required for an EDXS analysis, approximately 50 nanoCoulomb (50 nanoAmpere seconds), need not produce intolerable radiation damage when spread over an area 200 nm or more in diameter. Finer structure, for example mitochondria and regions of rough or smooth endoplasmic reticulum, can be identified and analysed in frozen-dried cryosections cut ～ 100 nm thick. Recently such features have been visualized in 100 nm frozen-hydrated sections where the water is vitreous. This opens the prospect of analysing material where elemental distributions have been preserved on a very fine scale, since one might avoid even the ionic shifts from aqueous solution to supramolecular structures which must occur on freeze-drying. But radiation damage may be prohibitive when an irradiation of 50 nanoCoulomb is concentrated into a hydrated area less than 200 nm in diameter.     

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.     

A nanostructured look of collagen apatite porosity into human mineralized collagen fibril

Bone tissue is a hierarchical material characterized at nanoscale by the mineralized collagen fibril, a recurring structure mainly composed of apatite minerals, collagen and water. Bone nanostructure has a fundamental role in determining the mechanical behavior of the tissue and its mass transport properties. Diffusion phenomenon allows to maintain an adequate supply of metabolites in the mechanisms of bone remodeling, adaptation and repair. Several analytical and computational models have been developed to analyze and predict bone tissue behavior. However, the fine replication of the natural tissue still represents a challenge. Insights on the structural organization at nanoscale and on the influence of apatite mineral crystals on the diffusion coefficient lead to outline the functional conditions for the development of biomimetic strategies for bone tissue engineering. Thorough understanding of bone nanostructure is essential to improve longevity of bioscaffolds and to decrease the risk of failure by controlling their mechanical and biological performance.     

Method of imaging low density lipoproteins by atomic force microscopy

This short paper reports a simple method to image low density lipoproteins (LDL) using atomic force microscopy (AFM). This instrument allows imaging of biological samples in liquid and presents the advantage of needing no sample preparation such as staining or fixation that may affect their general structure. Dimensions (diameter and height) of individual LDL particles were successfully measured. AFM imaging revealed that LDL have a quasi-spherical structure on the x and y axis with an oblate spheroid structure in the z axis (i.e., height). LDLs were found to have an average diameter of 23 +/- 3 nm. The obtained mean height was 10 +/- 2 nm.     

Technical advances in the sectioning of dental tissue and of on‐section cross‐linked collagen detection in mineralized teeth

Immunohistochemical detection of cross‐linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on‐section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New®. It was possible to cut thin (1 μm) sections of mineralized teeth, and immunofluorescence characterization of cross‐linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was “patchy” and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on‐section protein detection but importantly for detecting cross‐linked fibrous collagens in both soft and mineralized tissue sections. Microsc. Res. Tech. 73:741–745, 2010. © 2009 Wiley‐Liss, Inc.     

Effect of non-enzymatic glycation on collagen nanoscale mechanisms in diabetic and age-related bone fragility

Age and diabetes have long been known to induce an oxidative reaction between glucose and collagen, leading to the accumulation of advanced glycation end-products (AGEs) cross-links in collagenous tissues. More recently, AGEs content has been related to loss of bone quality, independent of bone mass, and increased fracture risk with aging and diabetes. Loss of bone quality is mostly attributed to changes in material properties, structural organization, or cellular remodeling. Though all these factors play a role in bone fragility disease, some common recurring patterns can be found between diabetic and age-related bone fragility. The main pattern we will discuss in this viewpoint is the increase of fibrillar collagen stiffness and loss of collagen-induced plasticity with AGE accumulation. This study focused on recent related experimental studies and discusses the correlation between fluorescent AGEs content at the molecular and fibrillar scales, collagen deformation mechanisms at the nanoscale, and resistance to bone fracture at the macroscale.     

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.