Ultrastructural and histochemical evaluation of appositional mineralization of circumpulpal dentin at the crown- and root-analog portions of rat incisors

Mineralization of circumpulpal dentin has been interpreted in such a way that predentin matrix is abruptly converted to almost fully mineralized dentin at the mineralization front. A group of investigators pointed out the existence of intermediary layer along the mineralization front of rat incisor dentin and claimed that dentin mineralization is a rather transient process. Owing to a paucity of information, however, the entity of transient mineralization of dentin has remained elusive. Here we confirmed the existence of a lightly mineralized layer (LL) along the mineralization front of rat incisor dentin, recognizable by both light and electron microscopy, in routinely processed specimens. LL less than 3?μm thick was shown to be located along the mineralization front of crown-analog dentin and tapered out toward the root analog of the incisor. Electron microscopy revealed that mineral deposition first occurred in the non-collagenous matrix of LL and that mineralization of collagen fibers took place sometime later at the conventional mineralization front. Microscopic appearance of the mineral phase of LL varied considerably depending on the histological processing of ultrathin sections, thus explaining the inconsistent interpretation of dentin mineralization in previous studies. These data suggest that mineralization of circumpulpal dentin in rat incisors proceeds in a stepwise or a transient manner, initiated by crystal deposition in the non-collagenous matrix followed by massive mineral deposition in collagen fibers at the mineralization front. The thickness of LL where only the non-collagenous matrix is mineralized may vary in relation to differences in the local non-collagenous matrix and also the rate of collagen mineralization in the respective portions of circumpulpal dentin.     

The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study

In a previous report we showed that young rats fed a calcium-free diet for 28 days developed severe hypocalcaemia and showed a significant increase in serum alkaline phosphatase activity. The main histological and cytochemical changes exhibited by these animals in bone of the metaphyseal primary spongiosa were: (1) hyperplasia of osteoblasts, (2) an increase in the frequency of tartrate-resistant acid phosphatase (TRAP)-positive osteoblasts apposed to osteoid, and (3) an excessive amount of osteoid tissue. In addition to typical osteoblasts, there was a subpopulation of osteoblast-like cells with coated pits, lysosome-like bodies and large cytoplasmic processes. In the present study, we investigated how the above parameters change when calcium-depleted rats are placed on a normal diet for 7 days. Such a regimen normalized calcium concentration and alkaline phosphatase activity in the serum. The osteoid thickness returned to normal and, in some areas, was fully calcified. Most osteoblasts no longer showed TRAP activity and their ultrastructure was similar to that found in controls. Despite an intense alkaline phosphatase activity, some of them still exhibited a number of macrophagic characteristics. They were TRAP-positive, and showed electron-dense bodies in the cytoplasm facing bone, an abundance of coated pits, calcified spicules impinging on the cell membrane and large processes extending into the mineralized matrix. We concluded that calcium deficiency causes hyperplasia of osteoblasts in primary spongiosa and an increase in expression of TRAP. It also induces changes in their phenotype characterized by the acquisition of macrophagic cellular features. While TRAP activity is normalized by calcium repletion, macrophagic characteristics persist. These results suggest that the osteoblast can modulate its phenotype according to its physiological status.     

Extracellular Matrix Control of Collagen Mineralization In Vitro

Collagen biomineralization is a complex process and the controlling factors at the molecular level are still not well understood. A particularly high level of spatial control over collagen mineralization is evident in the anchorage of teeth to the jawbone by the periodontal ligament. Here, unmineralized ligament collagen fibrils become mineralized at an extremely sharp mineralization front in the root of the tooth. A model of collagen biomineralization based on demineralized cryosections of mouse molars in the bone socket is presented. When exposed to metastable calcium and phosphate‐containing solutions, mineral re‐deposits selectively into the natively mineralized tissues with high fidelity, demonstrating that the extracellular matrix retains sufficient information to control the rate of mineralization at the tissue level. While solutions of simulated bodily fluid produce amorphous calcium phosphate within the tissue section, a more highly supersaturated solution stabilized with polyaspartic acid produces oriented, crystalline calcium phosphate with diffraction patterns consistent with hydroxyapatite. The model thus replicates both spatial control of mineral deposition, as well as the matrix‐mineral relationships of natively mineralized collagen fibrils, and can be used to elucidate roles of specific biomolecules in the highly controlled process of collagen biomineralization. This knowledge will be critical in the design of collagen‐based scaffolds for tissue engineering of hard‐soft tissue interfaces.     

Real electron microscopic images of collagen fibrils in the endoneurium

Collagen fibrils run in parallel in the endoneurial space, forming fibre bundles. Spaces are evident between these bundles when examined by transmission electron microscopy (TEM). However, the procedures for TEM include chemical fixation, dehydration and embedding, which may cause morphological changes in the specimens. Ultracryo thin sectioning procedures may avoid the artefacts caused by these procedures. An examination of ultrathin frozen sections revealed that the endoneurial space was completely filled with collagen fibrils, with little space between the fibre bundles. These results suggest that the dehydration and/or embedding procedures cause shrinkage of the specimen, resulting in the appearance of a widened space in the endoneurium. Therefore, the widened space between the bundles of collagen fibrils may be a technical artefact.     

Effects of enamel matrix derivative on mineralized tissue formation during bone wound healing in rat parietal bone defects

Enamel matrix derivative (EMD: Emdogain) has been reported to stimulate the biosynthesis and regeneration of trabecular bone. To address whether the biological action of EMD is dependent on the local environment of osseous tissue, circular perforations were made in parietal bones and immediately filled with either EMD or its carrier, propylene glycol alginate (PGA), as control. On post-operative days 4-60, the dissected bones were examined by various histological techniques. New bone matrix, which was immunoreactive for bone sialoprotein (BSP), was formed from the periosteum at the peripheral area of perforations. Different from the findings reported in injured long bones, mineralized tissue was produced in the regenerating connective tissue within bone defects. This mineralized tissue was hardly immunostained for BSP, contained few collagen fibres, and lacked osteocytic lacunae and layers of osteoblasts and osteoid. Energy-dispersive X-ray analysis showed that Ca and P weight % and Ca/P molar ratio of this mineralized tissue were similar to or slightly higher than those in the pre-existing parietal bones. In addition, most multinucleated cells located in mineralized tissue lacked a ruffled border structure and showed weak immunoreaction for the lysosomal cysteine proteinase, cathepsin K, whereas those located in the bone matrix exhibited ruffled borders and strong cathepsin K expression. However, multinucleated cells located in both tissues were strongly stained for tartrate-resistant acid phosphatase. The volume fraction of such mineralized tissue appeared to be higher in EMD-applied bones than in PGA-applied controls. The mineralized tissue-forming stromal cells within bone defects appeared to show greater accumulation in EMD-applied bones than in PGA-applied controls. Our results suggest that the bioactive effects of EMD on bone wound healing and mineralized tissue formation depend, at least in part, on the local osseous environment where EMD has been applied.     

Oriented Strontium Carbonate Nanocrystals within Collagen Films for Flexible Piezoelectric Sensors

Bone, assembled by mineralized collagen fibrils, displays piezoelectric properties under external stimulation to affect tissue growth. The mineralized collagen fibrils consist of collagen and oriented inorganic nanocrystals. Inspired from the unique structures and piezoelectric effect of mineralized collagen fibrils, the intrafibrillar mineralization of oriented strontium carbonate nanocrystals is achieved in vitro, which also exhibits good piezoelectric properties. The amorphous strontium carbonate precursors penetrate from the gap zones and fill gradually into the whole space within the collagen fibrils, and transform into a co-oriented crystalline phase. Isolated mineralized collagen fibrils with organized SrCO₃ nanocrystals acquire good flexible properties and inverse piezoelectric responses with an effective piezoelectric coefficient of 3.45 pm V⁻¹, much higher than individual collagen (1.12 pm V⁻¹) and SrCO₃ crystals (0.092 pm V⁻¹). These results may indicate that the organic and inorganic components synergistically contribute to the piezoelectric effect of bone. Furthermore, devices of flexible piezoelectric thin films assembled by SrCO₃ mineralized collagen fibrils exhibit a regular open-circuit voltage of 1.2 V under compressive stress and a stable cycling short-circuit current of 80 nA under a bending mode. It can also facilitate the development of promising piezoelectric sensors.     

An EDTA-KOH method to expose bone cells for scanning electron microscopy.

To observe bone cells by scanning electron microscopy (SEM), the mouse parietal bones were processed by decalcification with EDTA and digestion of collagen fibers with KOH to remove the bone matrix, in addition to the conventional preparation for SEM. The critical-point-dried specimens were split into two membranous pieces along the gaps formed by removing the bone matrix. By this method, osteoclasts showing full three-dimensional images of ruffled borders, osteoblasts showing special structures on the surfaces facing the bone matrix, and osteocytes extending many slender processes were clearly demonstrated in SEM. This new method may provide new viewpoints in bone cell biology.     

Structure and formation of the twisted plywood pattern of collagen fibrils in rat lamellar bone

This study was designed to elucidate details of the structure and formation process of the alternate lamellar pattern known to exist in lamellar bone. For this purpose, we examined basic internal lamellae in femurs of young rats by transmission and scanning electron microscopy, the latter employing two different macerations with NaOH at concentrations of 10 and 24%. Observations after the maceration with 10% NaOH showed that the regular and periodic rotation of collagen fibrils caused an alternation between two types of lamellae: one consisting of transversely and nearly transversely cut fibrils, and the other consisting of longitudinally and nearly longitudinally cut fibrils. This finding confirms the consistency of the twisted plywood model. The maceration method with 24% NaOH removed bone components other than cells, thus allowing for three-dimensional observations of osteoblast morphology. Osteoblasts extended finger-like processes paralleling the inner bone surface, and grouped in such a way that, within a group, the processes arranged in a similar direction. Transmission electron microscopy showed that newly deposited fibrils were arranged alongside these processes. For the formation of the alternating pattern, our findings suggest that: (1) osteoblasts control the collagen fibril arrangement through their finger-like process position; (2) osteoblasts behave similarly within a group; (3) osteoblasts move their processes synchronously and periodically to promote alternating different fibril orientation; and (4) this dynamic sequential deposition of fibrils results in the alternate lamellar (or twisted plywood) pattern.     

A 3D Cell-Free Bone Model Shows Collagen Mineralization is Driven and Controlled by the Matrix

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization, and different degrees of mineralization where the intrafibrillar and extrafibrillar minerals are intergrown into one continuous network of oriented crystals. While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix. Through the remineralization of demineralized bone matrix, the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement are demonstrated. Then, this cell-free in vitro system is explored as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Combined Raman and electron microscopy indicate that glycosaminoglycans (GAGs) play a more prominent role than generally assumed in the matrix–mineral interactions. The experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.     

Autologous Versatile Vesicles‐Incorporated Biomimetic Extracellular Matrix Induces Biomineralization

Restoring extracellular matrix (ECM) with supportive and osteoinductive abilities is of great significance for bone tissue regeneration. Current approaches involving cell‐based scaffolds or nanoparticle‐modified biomimic‐ECM have been met with additional biosafety concerns. Herein, the natural biomineralization process is first analyzed and is found that mesenchymal stem cells‐derived extracellular vesicles (EVs) from early and late stages of osteoinduction play different roles during the mineralization process. The functional EVs hierarchically with blood‐derived autohydrogel (AH) are then incorporated to form an osteoinductive biomimetic extracellular matrix (BECM). The alkaline phosphatase‐rich EVs are released from the outer layers to induce osteoblast differentiation during early stages. Thereafter, as the degradation of AH occurred, calcium/phosphorus (Ca/P)‐rich EVs are liberated to promote the nucleation of extracellular mineral crystals. Additionally, BECM contains considerable collagen fibrils that provide additional nucleation sites for crystallites deposition, thus reaching self‐mineralization in situ. In conclusion, this research provides a promising, versatile mineralization‐instructive platform to tackle the challenges faced in bone‐tissue engineering.     

Ultrahigh-resolution immunofluorescence microscopy using ultrathin cryosections: subcellular distribution of caveolin-1alpha and CD31 in human placental endothelial cells

We compared the z-axis resolutions achieved by immunofluorescence (IF) microscopic imaging of tissue sections of different thicknesses (ultrathin cryosections, optical sections of cryostat sections and conventional cryostat sections). We used these images to determine the distribution of caveolin-1alpha (CAV-1alpha) and CD31 in endothelial cells of full-term, human placenta. Anti-CAV-1alpha antibody was used to visualize caveolae, which are among the smallest organelles. By using ultrathin cryosections as substrates for IF microscopy, we were able to resolve discrete caveolae that were primarily present immediately beneath the endothelial cell surface. In contrast, neither conventional nor confocal images from cryostat sections were able to resolve individual caveolae, despite dramatic reductions in the confocal image degradation that arises from out-of-focus fluorescence signals. Anti-CD31 antibody labeled the endothelial cell surface exclusively. Quantitative analysis of ultrathin cryosections showed that about 2.5 times more CD31 was expressed on the luminal surface of cells than on the abluminal surface. Our results demonstrate that ultrathin cryosections can serve as excellent substrates for ultrahigh-resolution IF microscopy.     

透射电镜神经组织制样包埋剂比较

为探讨两种常用包埋剂Epon 812和Eponate 12对神经组织透射电镜样品的影响,本文采用多聚甲醛-戊二醛溶液对wistar大鼠进行全身灌流固定后,取材海马、脊髓及坐骨神经,按常规透射电镜样品技术制作超薄切片.电镜观察显示,经Eponate 12包埋后的样品,在切片的透明度及超微结构的清晰度上均优于Epon 812包埋的超薄切片样品.由于Eponate 12的浸透效果好,有效地避免了有髓神经纤维髓鞘上出现孔洞结构的制样问题.本实验小组的实验结果表明:对于神经组织,尤其是周围神经组织的超薄切片样品,使用Eponate 12作为包埋剂,效果会更好一些.     

Matrix remodeling and cytological changes during spontaneous cartilage repair

During the repair of articular cartilage, type I collagen (COL1)-based fibrous tissues change into a mixture of COL1 and type II collagen (COL2) and finally form hyaline cartilaginous tissues consisting of COL2. In order to elucidate the changes that occur in the matrix during cartilage repair and the roles of fibroblasts and chondrocytes in this process, we generated a minimal cartilage defect model that could be spontaneously repaired. Defects of 0.3?mm were created on the patellofemoral articular cartilage of rats using an Er:YAG laser and were observed histologically, ultrastructurally and histochemically. At week 2 after this operation, fibroblastic cells were found to be surrounded by COL1 throughout the area of the defect. These cells became acid phosphatase positive by week 4, both taking in and degrading collagen fibrils. Thereafter, the cells became rounded, with both COL1 and 2 evident in the matrix, and showed immunolocalized matrix metalloproteinase-1 or -9. In the region of the bone marrow, the cells became hypertrophic and were surrounded mainly by COL2 and proteoglycans. By the eighth week, the cartilaginous matrix was found to contain abundant COL2, in which collagen fibrils of various diameters were arranged irregularly. These morphological changes suggested that the fibroblastic cells both produce and resolve the matrix and undertake remodeling to become chondrocytes by converting from a COL1- into a COL2-dominant matrix. This process eventually forms new articular cartilage, but this is not completely identical to normal articular cartilage at the ultrastructural level.     

Correlative light microscopy, scanning electron microscopy, and transmission electron microscopy of osmium-macerated biological tissues

A method facilitating correlation of light microscopic (LM), scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images was developed. Rat kidney and heart were initially subjected to the osmium maceration procedure and then embedded in acrylic resin. Semithin sections of the tissue blocks were first provided for LM and then examined by SEM after resin removal. Furthermore, the ultrathin sections adjacent to the semithin sections were observed by TEM. The three-dimensional images of intracellular organelles provided an informative adjunct to LM and TEM.     

Organic components of crystal sheaths in bones

Crystals in bones are enveloped within organic crystal sheaths of 5-10 nm widths. In order to analyse their components, we investigated the immunolocalizations of chondroitin 4- and 6-sulphate, keratan sulphate, bone sialoprotein and osteopontin. All of these, except chondroitin 6-sulphate, were found in bone matrix. Although the localizations of chondroitin 4-sulphate and keratan sulphate tended to focus within calcified nodules, bone sialoprotein and osteopontin were widely distributed in the area, being linearly arranged along electron-dense structures corresponding to crystal sheaths. These two proteins possess the ability to affect nucleation or elongation of hydroxyapatite, positively or negatively, in vitro. Our results suggested that bone sialoprotein and osteopontin may combine to form the crystal sheaths which are thought to control crystal formation and growth, using the seemingly opposite functions of bone sialoprotein and osteopontin.     

Measurement of the repeat period of myelin sheath using ultrathin frozen sections

The myelin sheath of peripheral nerves was observed by transmission electron microscopy (TEM) using plastic-embedded sections and ultrathin frozen sections. Repeat distances of myelin sheaths were measured in high-powered electron micrographs. The ultrathin frozen sections showed a longer repeat distance than the plastic-embedded sections. The ultrathin frozen sections were thought to contain fewer artefacts, as they had not been subject to dehydration and embedding. It is known that broken myelin sheaths are often observed under conventional TEM. It is thought that these procedures cause contraction and partial destruction of the myelin sheath.     

Statistical analysis of collagen alignment in ligaments byscale-space analysis

Injuries to ligaments in the knee are common in sports and other physical activities. Some clinical methods are available for qualitatively evaluating the degree of ligament injury and healing. It is, however, desirable to objectively assess the healing of ligaments and to predicate optimal treatment on quantitative measurements of their structure. Information such as areas of coverage and spatial orientations of collagen fibrils, for example, may provide important information about the internal structure of ligament tissues. Since normal ligament tissues are made up of collagen fibrils which are highly organized, they can be considered as oriented piecewise linear patterns. In this paper, we propose a computational technique for statistical analysis of collagen alignment in ligament images using the scale-space approach. In this method, a ligament image is preprocessed by a sequence of filters which are second derivatives of two-dimensional Gaussian functions with different scales. This gives a set of zero-crossing maps (the scale space) from which a stability map is generated. Significant linear patterns are captured by analyzing the stability map. The directional information in terms of orientation distributions of the collagen fibrils in the image and the area covered by the fibrils in specific directions are extracted for statistical analysis. Examples illustrating the performance of this method with scanning electron microscope images of the collagen fibrils in healing rabbit medial collateral ligaments are presented in this paper.     

The three-dimensional structure of the clear zone of a cultured osteoclast

Osteoclasts collected from long bones of newborn mice were cultured on dentine slices. Then, osteoclasts were sectioned by alternating semithin and ultrathin sections, and the three-dimensional reconstruction was made by the serial semithin sections. By this method, the present study showed first the three-dimensional structure of an osteoclast, especially the clear zone. A reconstructed osteoclast with complicated contours shifted from the lacuna, and its clear zone was a ring-shaped structure. By TEM, a new small lacuna was formed under the ruffled border, and the clear zone could be further classified into three types. The present study suggested that the synthetic observations of both three-dimensional structure and ultrastructure by transmission electron microscopy were necessary to determine whether the osteoclast was resorbing or migrating. According to these observations, the reconstructed osteoclast seemed to be migrating.     

Some observations on the subfibrillar structure of collagen fibrils as noted during treatment with NKISK and cathepsin G with mechanical agitation

We observed the structure of collagen fibrils in rat tail tendons after treatment with NKISK and cathepsin G. NKISK is a pentapeptide that has been previously shown to bind fibronectin, while cathepsin G is a serine protease that cleaves fibronectin but not type I collagen. In tendons treated with NKISK, fibrils were seen to extensively dissociate into smaller-diameter subfibrils. These subfibrils were homogeneous in diameter with an average diameter of 26.3?±?5.8?nm. Similar, although less extensive, dissociation into subfibrils was found in tendons treated with cathepsin G. The average diameter of these subfibrils was 24.8?±?4.9?nm. The ability of NKISK and cathepsin G to release subfibrils at physiological pH without harsh denaturants may enhance the study of the subfibrillar structure of collagen fibrils.     

Ultrastructural and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans

Histochemical, immunohistochemical and electron energy-loss spectroscopic studies were performed to examine the relationship between sulphated glycosaminoglycans and medullary bone calcification using oestrogen-injected male Japanese quail. Sulphated glycosaminoglycans, detected by high iron diamine (HID) or HID-thiocarbohydrazide-silver protein (HID-TCH-SP) methods, were distributed throughout the matrix of medullary bone, some periphery and extending tips of the trabeculae stained weakly, and the globular structures at osteoid areas were exclusively positive for HID-TCH-SP stain. Immunohistochemistry identified keratan sulphate located in the globular structures at osteoid areas and calcified matrix, but chondroitin-4 sulphate and chondroitin-6 sulphate were not detected in the matrix. Using electron spectroscopic imaging, sulphur was determined to be localized in the globular structures. These results demonstrate that medullary bone matrix accumulates keratan sulphate in the globular structures, which are the foci for calcification, and eventually in the calcified areas. This suggests that keratan sulphate containing sulphur is maintained in the calcified matrix. These results indicate a unique process of calcification exists in medullary bone.