Cryopreservation and image enhancement of juvenile and adult dentine mineral

The inorganic component of bone and related hard tissues is generally described as sheets of uniform needle- and plate-like crystals. However, cryofixation has become the method of choice for ultrastructural studies of bone mineral when ladder-like arrangements of filaments contained within deformable microspheres about 1µm in diameter are apparently the prime structural feature and are consistent with the optical image. The same methodology has now been applied to mature human dentine in caries-free juvenile and adult teeth. These were fixed, sliced, stained for mineral and examined optically or were snap frozen, fragmented under liquid nitrogen, freeze-substituted with methanol or acetone and embedded without thawing in Lowicryl K4M for electron microscopy. Others were processed by traditional transmission electron microscopy methods. To obtain maximum resolution, the electron micrographs were photographically printed as negatives and image-enhanced by digitisation using a Polaroid Sprint Scan 45 and laser printer. In both optical and cryopreparations of juvenile and adult dentine, mineral microspheres up to 1µm in diameter, were present in the dentinal tubules and peritubular dentine. Within these objects, the mineral was primarily in the form of sinuous electron dense filaments, 5nm thick, which had a characteristic periodicity. In these preparations needle-like and plate-like structures were rare. In contrast, after traditional transmission electron microscopy preparation although similar filamentous structures remained, the mineral more generally had the familiar form of needles measuring approximately 50nm in the long axis. The cryopreserved calcified filaments were apparently particularly densely distributed in the intertubular dentine where their parallel ladder-like arrays often formed highly orientated struts and stays. It was concluded that early dentine mineral has the form of filamentous microspheres and as in bone (and other calcifying tissues and cells) has no specific association with collagen. It was also concluded that these structures compact and deform with maturity into a sub-structural framework which may relate to powerful biomechanical forces transmitted through the tissue. Needle- or plate-like mineral is probably rare in vivo in dentine, only becoming commonplace after extensive chemical processing.     

Comparative study of the ESR spectra of normal and regenerating liver tissue in mice

Low temperature ESR spectra of gamma-irradiated samples of mouse liver regenerating after partial hepatectomy were studied. As compared to ESR spectra of gamma-irradiated samples of health animals liver, in regenerating liver samples new ESR signals from paramagnetic centres S(2.0005) and S(2.0025) were revealed; these signals were earlier recorded by the authors in gamma-irradiated samples of hepatoma 22a. It is concluded that the paramagnetic centres S(2.0005) and S(2.0025) are typical of proliferating tissues of different ethiology.     

Evidence for Phosphoprotein Microspheres in Bone

Bone sialoprotein and osteopontin are bone-specific phosphoproteins, but their function is uncertain and their ultrastructural associations remain unclear. Insight into their role was sought by special attention to their general distribution and specific morphology under the high-power optical microscope. Their extracellular staining characteristics were examined in cryosections of adult rat skeletal tissues using two immunohistochemical methods. The two proteins were clearly evident in immature woven bone of endochondral and intramembranous origin (although cartilage was negative, even when calcified). In mature lamellar bone, bone sialoprotein remained ubiquitous, while osteopontin was confined to cement lines and other relatively discrete sites of past and present resorption activity, particularly near blood vessels. In neither case was the distribution of the stain structureless and diffuse. Invariably (except when non-specific), it was sharply defined and had the form of microspheres measuring approximately 1 m in diameter. In both immature and mature regions, these objects appeared in sheets, chains or groups in a pattern that was evidently coincident with a similar structural arrangement found within the inorganic phase of bone. It was concluded that phosphoproteins are not randomly located throughout the collagenous matrix but are apparently integral to calcified microsphere populations, and it is suggested that these structures are well placed to control the chemical State of the mineral over their surfaces and influence remodelling.     

RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

Introduction

The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.

Methods

Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E₂ (PGE₂), then further stained with tartrate-resistant acid phosphatase.

Results

Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE₂-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.

Conclusions

Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.     

Applications of ion beam analysis to calcified tissue research

Various nondestructive ion beam analysis techniques have been developed and applied to study the concentrations of fluorine and other trace elements in calcified tissues. Fluorine has been determined by prompt gamma activation analysis through the F(?,αγ)O reaction. This method is quick, convenient, and sensitive, and can also be applied to measure fluorine depth distribution nondestructively in teeth and bone samples. By the application of this technique, fluorine concentrations have been determined in a number of teeth with known histories and bones of experimental animals. Moreover, F-depth-distributions to 10-μm depths have also been obtained in a number of selected human and animal teeth. Carbon was measured by activation analysis with He-3 ions, and the technique of Proton-Induced X-ray Emission (PIXE) has been developed to simultaneously determine Ca, P, and trace elements in well-documented teeth. Dental hard tissues, i.e., enamel, dentine, cementum, and their junctions, as well as different parts of the same tissue, have been examined separately. A number of elements, Na, Mg, Al, P, Cl, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Sr, Br, Rb and Pb, have been determined in these dental tissues. The concentrations of some of these elements vary considerably in different teeth and in various parts of the same tooth. The special advantages of these nuclear techniques for studying metabolic bone diseases, renal diseases, and the effect of Pb on childrens' intelligence are discussed.     

Fibroblastic cells from human periapical granulation tissue preferentially form calcified matrices in decalcified boiled rat bone

We have been studying the potential of human fibroblastic cells (HFC) from periapical granulation tissue to form a calcified matrix. Recently, we reported that inflamed periapical granulation tissue contains osteogenic cells. In the present study, we tested the hypothesis that HFC, cultured with decalcified bone (DB) of rat, might form much greater calcified matrices than with rat decalcified boiled bone (DBB), which was originally prepared as a negative control. HFC were cultured with DB or DBB in the presence or absence of 2 mM -glycerophosphate (-GP) and 50 g/ml ascorbic acid. After six weeks of culture, a number of von Kossa-positive globular structures were unexpectedly observed inside DBB, but not DB. Without HFC, such structures were never seen in DBB incubated with 2 mM -GP and 50 g/ml ascorbic acid. DB cultured with HFC under the same conditions did not show these structures. Electron-microscopic observation revealed that matrix vesicles aggregated on collagen fibrils around globular structures in DBB. Energy dispersive X-ray microanalysis confirmed that these structures were calcified matrices composed of calcium and phosphate. These results suggest that human periapical granulation tissue contains cells responsible for the formation of calcified matrices in DBB, and that DBB could serve as an excellent scaffold for the calcification of HFC, rather than DB.This work was supported by grant-in-aid (project 15689024) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.     

Role of particle coating in controlling skin damage photoinduced by titania nanoparticles

TiO₂ nanoparticles hazard is associated to their photocatalytic activity causing release of DNA damaging ROS (Reactive Oxygen Species), lipid peroxidation and skin damage. Various coatings have been proposed to minimize photocatalysis, while keeping the potential to block UV radiations. Uncoated and variously coated commercial nano-titania have been classified on the basis of UVB-induced lipoperoxidation of linoleic acid. A selection of the most and the least protective specimens was then examined by ESR (Electron Spin Resonance) to evidence the presence of surface paramagnetic centres and the release of ROS in aqueous suspensions (spin trapping). Paramagnetic centres and ROS were correlated with the extent of lipid peroxidation. When tested on porcine skin (mimicking the human one), titania acted as on linoleic acid. The combined use of lipid peroxidation of simple fatty acids with ESR analysis is here proposed as a possible screening tool for the evaluation of the potential toxicity of nano-titania in sunscreen preparations.     

Content and Distribution of Noncollagenous Matrix Proteins in Bone and Cementum: Relationship to Speed of Formation and Collagen Packing Density

The organic matrix of collagen-based calcified tissues consists of a supporting collagen meshwork and various noncollagenous matrix proteins (NCPs). Together, they contribute to determining the structure and biomechanical properties of the tissue. Their respective organization and interrelation can advantageously be examined by immunocytochemistry, an approach which allows correlation of composition with structure. The aim of this article is to review postembedding immuno- and lectin-gold-labeling data on the characterization of the noncollagenous compartment in rat and human bone and cementum, and on its relationship to collagen. The two major NCPs, bone sialoprotein and osteopontin, generally codistribute and accumulate in cement lines and in the spaces among the mineralized collagen fibrils. However, there are variations in their distribution and density of labeling throughout the tissue. Indeed, bone and cementum can form in environments that are either poor or enriched in NCPs. The amount of NCPs generally correlates with bone and cementum types and with speed of formation of the tissue and packing density of collagen fibrils. Taken together, the data suggest that production of both collagenous and noncollagenous constituents can be "modulated" during formation of collagen-based calcified tissues. It is concluded that, in addition to structural and compositional parameters, tissue dynamics must be taken into consideration in order to understand the significance of the apparent accumulation of NCPs at some sites and to determine the mechanisms of normal and pathological calcified tissue formation.     

Electron Spin Resonance (ESR) spectrum of paraffin embedded human liver tissue

Summary Human liver tissues embedded in paraffin wax for histological examination have been studied by Electron Spin Resonance (ESR) spectroscopy. A signal was detected at g2.05 section of the spectrum. The amplitude of this signal was correlated with the copper content of the embedded specimens measured by flame atomic absorption technique. The positive correlation which has been found can make ESR spectroscopy suitable for estimating the copper content of tissues without damaging the sample. The limits and errors of this method have also bee analysed.     

Detection of new metabolic paramagnetic centers in the lung tissue by ESR method

An ESR signal with g = 2.0025 and delta H = 0.2 mT was recorded at 77 K in lung tissues of guinea-pigs, mouse and rats. The signal intensity changed in the lung tissues of animals exposed to some chemicals. A relationship between new paramagnetic centres and the lung surfactant system is suggested to exist.     

The effect of T-2 toxin on bone microstructure in rabbits

T-2 toxin is the most toxic of the trichothecene mycotoxins. Its effect on bone microstructure is still unknown. This study focuses on acute effects of the T-2 toxin on compact and trabecular bone tissues structure of rabbits after a single intramuscular administration. Experimental E group (n?=?4) consisted of animals which were intramuscularly injected with T-2 toxin at dose 0.08 mg.kg^?1 body weight 72 h before slaughter. Group C (n?=?4) without T-2 toxin application served as a control. An absence of primary vascular longitudinal bone tissue near endosteal surfaces, its deposition on periosteal surfaces and a lower density of secondary osteons in the middle part of the substantia compacta were observed in both females and males injected with T-2 toxin. On the contrary, morphometrical analysis of the compact bone showed no demonstrable alternations in the sizes of primary osteons’ vascular canals, Haversian canals or secondary osteons between rabbits from E and C groups. Also, no significant effects of the T-2 toxin on trabecular bone morphometry and cortical bone thickness were observed between rabbits of either sex. The single intramuscular application of T-2 toxin at the dose used in our study affects only qualitative histological characteristics of the compact bone in rabbits.     

Computer simulation of ESR signals of liver paramagnetic centers

Changes of paramagnetic centres concentration characterized by g-factors values of 1.94, 2.2, and 2.03 in the rat liver were studied by ESR method under acute intoxication by diethylnitrosamine (DENA) and at preliminary threefold treatment of animals with butylhydroxytoluene (BHT). A protective effect of BHT can be explained by its stabilizing action of the membrane structures. A comparison has been carried out with a similar study of paramagnetic centres in the experiment of chronic intoxication by DENA. A simulation was performed of the liver tissue ESR spectra by means of special computer program. The parameters of simulated ESR spectra of the liver tissue with due regard for ESR signal g 2.03 corresponded to the parameters of the experimental spectra. Confirmations were obtained for the nature and number of paramagnetic centres in the liver tissue.     

Effect of gamma radiation and accelerated electron beam on stable paramagnetic centers induction in bone mineral: influence of dose,irradiation temperature and bone defatting

Ionizing radiation has been found to induce stable defects in the crystalline lattice of bone mineral hydroxyapatite, defined as CO₂ ^? radical ions possessing spins. The purpose of our study was to evaluate CO₂ ^? radical ions induced in non-defatted or defatted human compact bone by gamma radiation (G) and accelerated electron beam (EB), applied with two doses at different temperatures. Moreover, the potential effect of free radical ion formation on mechanical parameters of compact bone, tested under compression in the previous studies, was evaluated. Bone rings from femoral shafts of six male donors (age 51 ± 3 years) were collected and assigned to sixteen experimental groups according to different processing methods (non-defatted or defatted), G and EB irradiation dose (25 or 35 kGy), and irradiation temperature [ambient temperature (AT) or dry ice (DI)]. Untreated group served as control. Following grinding under LN₂ and lyophilization, CO₂ ^? radical ions in bone powder were measured by electron paramagnetic resonance spectrometry. We have found that irradiation of bone with G and EB induces formation of enormous amounts of CO₂ ^? radical ions, absent from native tissue. Free radical ion formation was dose-dependent when irradiation was performed at AT, and significantly lower in EB as compared to G-irradiated groups. In contrast, no marked effect of dose was observed when deep-frozen (DI) bone samples were irradiated with G or EB, and free radical ion numbers seemed to be slightly higher in EB-irradiated groups. Irradiation at AT induced much higher quantities of CO₂ ^? radical ions then on DI. That effect was more pronounced in G-irradiated bone specimens, probably due to longer exposure time. Similarly, bone defatting protective effect on free radical ion formation was found only in groups irradiated for several hours with gamma radiation at ambient temperature. Ambient irradiation temperature together with exposure time seem to be key parameters promoting CO₂ ^? radical ion formation in bone mineral and may mask the opposite effect of defatting and the possible effect of irradiation type. Significant weak negative correlations between CO₂ ^? radical ion number and some mechanical properties of compact bone rings (Young’s modulus and ultimate stress) were found.     

Microradiographic study of prehistoric skeletal remains

Microradiography is a standard technique for determining small differences in mineral content in histological sections of calcified tissues by using soft X-rays. Such differential mineralization points on physiological and pathological processes in bone and teeth, and is frequently conserved both in excavated skeletal remains and in cremations. Continuing from this, microradiography gives diagnostic criteria for histological and palaeopathological investigations on historic and prehistoric skeletal remains rather than ordinary light microscopy. In addition, this technique helps in reconstructing the genesis of special dead bone decomposition phenomena.     

Preferential activation of SMAD1/5/8 on the fibrosa endothelium in calcified human aortic valves--association with low BMP antagonists and SMAD6

Background

Aortic valve (AV) calcification preferentially occurs on the fibrosa side while the ventricularis side remains relatively unaffected. Here, we tested the hypothesis that side-dependent activation of bone morphogenic protein (BMP) pathway in the endothelium of the ventricularis and fibrosa is associated with human AV calcification.

Methods and Results

Human calcified AVs obtained from AV replacement surgeries and non-calcified AVs from heart transplantations were used for immunohistochemical studies. We found SMAD-1/5/8 phosphorylation (a canonical BMP pathway) was higher in the calcified fibrosa than the non-calcified fibrosa while SMAD-2/3 phosphorylation (a canonical TGFβ pathway) did not show any difference. Interestingly, we found that BMP-2/4/6 expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both calcified and non-calcified AV cusps; however, BMP antagonists (crossvienless-2/BMPER and noggin) expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both disease states. Moreover, significant expression of inhibitory SMAD-6 expression was found only in the non-calcified ventricularis endothelium.

Conclusions

SMAD-1/5/8 is preferentially activated in the calcified fibrosa endothelium of human AVs and it correlates with low expression of BMP antagonists and inhibitory SMAD6. These results suggest a dominant role of BMP antagonists in the side-dependent calcification of human AVs.     

Association between Randall's plaque and calcifying nanoparticles

Objectives

Randall initially described calcified subepithelial papillary plaques, which he hypothesized as nidi for urinary calculi. The discovery of calcifying nanoparticles (CNP), also referred to as nanobacteria, in calcified soft tissues has raised another hypothesis about their possible involvement in urinary stone formation. This research is the first attempt to investigate the potential association of these two hypotheses.

Methods

We collected renal papilla and blood samples from 17 human patients who had undergone laparoscopic nephrectomy. Immunohistochemical staining (IHS) was applied using monoclonal antibody (mAb) against CNP. Homogenized papillary tissues and serum samples were cultured for CNP. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) were performed on papillary samples. Serum samples were tested for CNP antigen and antibody with enzyme-linked immunosorbent assay (ELISA).

Results

Randall’s plaques (RP) were visible on gross inspection in 11 out of 17 samples. IHS was positive for CNP antigen in 8 of the visually positive samples, but in only 1 of the remaining samples. SEM revealed spherical apatite-formations in 14 samples confirmed by EDS analysis. In cultures, all serum samples and 13 tissue homogenates grew CNP. In ELISA, 14 samples were positive for CNP-antigen and 11 samples were positive for CNP-antibody.

Conclusion

There was evidence of a link between detection of CNP and presence of RP. Although causality was not demonstrated, these results suggest that further studies with negative control samples should be made to explore the etiology of RP formation, thus leading to a better understanding of the pathogenesis of stone formation.     

A library of mammalian effector modules for synthetic morphology

Background

In mammalian development, the formation of most tissues is achieved by a relatively small repertoire of basic morphogenetic events (e.g. cell adhesion, locomotion, apoptosis, etc.), permutated in various sequences to form different tissues. Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms. The control of tissue morphogenesis by populations of engineered cells is a potentially very powerful but neglected aspect of synthetic biology.

Results

We have assembled a modular library of synthetic morphogenetic driver genes to control (separately) mammalian cell adhesion, locomotion, fusion, proliferation and elective cell death. Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command.

Conclusions

Building on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.