Porcine enamel matrix derivative enhances trabecular bone regeneration during wound healing of injured rat femur.

To elucidate the effects of enamel matrix derivative (EMD: Emdogain) on bone regeneration in rat femurs after drill-hole injury, defects in bone were filled with either EMD or its carrier, PGA, as control. On postoperative days 4 to 28, dissected femurs were examined by means of various morphological approaches. In both experimental groups, formation of trabecular bone, which was immunostained for bone sialoproteins (BSP), had occurred in the medullary cavities at cylindrical bone defects on Day 7 postoperatively. Cuboidal osteoblasts were clearly observed on these newly-formed BSP-positive bone trabeculae. On Days 7 and 14, many multinucleated giant cells, which strongly expressed cathepsin K, had appeared on these bone trabeculae, indicating active bone remodeling. In these bone trabeculae, Ca and P weight % and Ca/P ratio were similar to those of cortical bone, and there was no significant difference between the PGA- and EMD-applied groups. Bone volume fraction of newly-formed bone trabeculae on Day 7 postoperatively was significantly higher in the EMD-applied group than in the PGA-applied controls. Because of active bone remodeling and the marked decrease of bone volume, on Days 14 and 28 postoperatively, however, there was no longer a significant difference in trabecular bone volume fraction between the experimental groups. Our results suggest that EMD possesses an osteo-promotive effect on bone and medullary regeneration during wound healing of injured long bones.

Acute effects of ovariectomy on wound healing of alveolar bone after maxillary molar extraction in aged rats.

Acute effects of ovariectomy on the bone wound healing processes after maxillary molar extraction in aged rats were examined by means of quantitative scanning electron microscopy (SEM), backscattered electron image (BSE) analysis and energy-dispersive X-ray (EDX) microanalysis. Six-month-old female rats underwent either sham operation or bilateral ovariectomy, and 7 days postoperatively, the maxillary first molars were extracted. On post-extraction days 7, 30 and 60, the dissected maxillary bone surfaces were examined by SEM to reveal the bone formative and resorptive areas around the extracted alveolar sockets. In addition, the resin-embedded maxillae were micromilled in the transverse direction through the extracted alveolar sockets, and the newly-formed bone mass on the buccal bone surfaces and within the extracted sockets was examined by BSE analysis. Compared with sham-operated controls, the extent of newly-formed bone mass on the buccal bone surfaces in OVX rats was significantly decreased, due to increased bone resorption. On the other hand, new bone formation within the extracted sockets was similar in the experimental groups. In EDX microanalysis of these newly-formed bone matrices, both Ca and P weight % and Ca/P molar ratio were similar in the experimental groups. Our results suggest that 1) acute estrogen deficiency induced by ovariectomy stimulates sustained bone resorption, but has less effect on bone formation, and 2) bone wound healing after maxillary molar extraction within extracted alveolar sockets is not significantly delayed by ovariectomy, but bony support by newly-formed bone mass on the maxillary bone surfaces at the buccal side of the extracted sockets is significantly decreased, due to increased bone resorption.

Effects of ovariectomy on bone morphology in maxillae of mature rats.

Postmenopausal oestrogen deficiency results in bone loss (osteoporosis) in humans and experimental animals. The loss of trabecular bone in the ovariectomized (OVX) rat provides a useful experimental model of post-menopausal osteoporosis. At 5 months after ovariectomy of 3-month-old female rats, the mid and distal femurs and maxillae were dissected and processed for quantitative backscattered electron microscopic examinations. Histomorphometric analysis of femurs in OVX rats showed significant loss in metaphyseal trabecular bone areas compared with sham-operated controls; no significant bone loss was observed in the cortical bone areas of mid-diaphyses in OVX rats. Net bone areas in the maxillae of OVX rats was similar to that of sham-operated controls. Bone structure of maxillae in OVX rats was also similar to that in controls. Our results suggest that, in this animal model of osteoporosis, prominent bone loss occurs mainly in the bone areas formed by endochondral ossification such as distal femurs, but those areas formed by intramembranous ossification such as mid-femurs and maxillae sustained less effects by OVX.

Effects of chemically modified tetracycline, CMT-8, on bone loss and osteoclast structure and function in osteoporotic states.

We examined the effects of a nonantimicrobial tetracycline analogue, CMT-8, on bone loss and osteoclasts in ovariectomized (OVX) rats. Three-month-old female rats were OVX, and, one week later, distributed into three groups: sham-operated non-OVX controls, untreated OVX controls, and CMT-8-treated OVX rats. After 145 days of daily drug administration (p.o.), the femurs were dissected and examined histologically. Ovariectomy markedly decreased trabecular and cortical bone volume in the metaphyses compared to sham-operated controls. Treating the OVX rats with CMT-8 produced a significant inhibition of trabecular and cortical bone loss and induced new bone formation, in which connectivity of the trabecular struts was increased by bridging the adjacent longitudinal bone trabeculae. Ultrastructurally, CMT-8 reduced ruffled border formation in osteoclasts, while it caused no structural impairment in osteoblasts. To further evaluate the effects of CMT-8 on the resorbing activity of osteoclasts, osteoclasts were cultured on dentine slices pretreated with CMT-8 at concentrations of 2, 10, or 50 micrograms/ml, and resorption lacuna formation on the dentine surface was found to be reduced, dose-dependently, by the bound CMT-8. Our results suggest that CMT-8 therapy effectively inhibits post-ovariectomy bone loss not only by inducing new bone formation, but also by inhibiting osteoclastic bone resorption, and that CMT-8 binding to bone may provide a prolonged release delivery of this anti-resorptive therapy.

Cartilage-bone replacement in endochondral ossification of mandibular condylar heads in young beagle dogs.

The cellular mechanisms of cartilage-bone replacement in endochondral bone formation, in mandibular condylar heads, are poorly understood. In particular, there is no definitive evidence indicating whether cartilage is resorbed by so-called chondroclasts. Using 3-week-old male beagle dogs, we examined the cartilage-bone replacement processes in mandibular condylar heads by means of light and electron microscopy. Calcification of the cartilage matrix occurred in the central area of the longitudinal septa but not in thin transverse septa. Chondrocytic lacunae were opened by the removal of transverse septa by perivascular rough-surfaced endoplasmic reticulum (RER)-rich mononuclear cells. These cells also phagocytosed calcified cartilage fragments in the surface layer of longitudinal septa. Shortly thereafter, a thin bone layer was deposited on the remaining longitudinal septa by invading osteoblasts. Preosteoclastic multinucleated cells in lacunar canals developed neither ruffled borders nor clear zones in the cartilage matrix, but once the bone layer had been deposited on the remaining cartilage, these structures formed. Our results suggest that the cartilage-bone replacement in mandibular condylar heads involves four sequential processes: 1) degradation of the transverse septal cartilage by RER-rich mononuclear cells, 2) phagocytosis of calcified cartilage fragments in the longitudinal septa by these cells, 3) bone deposition of the remaining longitudinal septa, and 4) degradation of both bone and calcified cartilage by differentiated osteoclasts.

[The replacement processes of growth plate cartilage to bone in endochondral ossification of mandibular condyle].

Using 3-week-old male beagle dogs, we examined the cartilage-bone replacement processes in the mandibular condyle by means of light and electron microscopy. Calcification of the cartilage matrix occurred in the central area of the longitudinal septa, but not in the transverse septa. Perivascular mononuclear cells absorbed the transverse septa which initiated the opening of the chondrocytic lacunae. These cells phagocytosed septal cartilage fragments. Shortly thereafter, a thin bone layer was deposited on the remaining longitudinal septa by invading osteoblasts. Osteoclasts in lacunae developed neither ruffled borders nor clear zones in the cartilage matrix, but once the bone layer has been deposited in the remaining cartilage, these structures formed. Our results suggest that the cartilage-bone replacement in mandibular condyle involves three sequential processes: 1) degradation and phagocytosis of cartilage fragments in the transverse septa by mononuclear cells, 2) bone deposition over the remaining longitudinal septa, and 3) degradation of both calcified cartilage and bone by osteoclasts.

Energy-dispersive X-ray microanalysis of calcified tissues after NaOCl treatment and/or resin embedding.

By energy-dispersive X-ray microanalysis (EDX) in scanning electron microscopy, we studied the calcification of the inner-basic lamellas of a cow bone and the coronal cementum of a horse tooth treated with sodium hypochlorite (NaOCl). These tissues were divided into 4 groups with a combination of NaOCl treatment and polyester-resin embedding including, [A]: non-NaOCl + Resin, [B]: non-NaOCl + non-resin, [C]: NaOCl + Resin, and [D]: NaOCl + non-Resin. From the Ca and P values by EDX analysis, it was suggested that the natural porous spaces of [B] were higher than those of [A], and both the natural and NaOCl-soluble porous spaces were highest in [D]. However, [A] had the lowest porous spaces in both the tissues because the micropores formed 3-dimensionally by NaOCl treatment might be incompletely filled with the resin. The backscattered electron microscopy and the difference of the Ca/P ratios indicated that the NaOCl treatment of the calcified tissues caused some minerals besides organic materials to dissolve. Thus, the sample preparation of [B] is suitable for the quantitative EDX of calcified tissues, whereas the data of [C] except for the Ca/P ratio may be used to approximate the Ca and P contents.

Backscattered electron imaging and energy-dispersive X-ray microanalysis studies of evidence for calcium salt heterogeneity in fifteen gallstones from an elderly human.

We examined 15 variably-sized gallstones, taken from an elderly male, by backscattered electron imaging and energy-dispersive X-ray microanalysis to learn the structural and distribution patterns of gallstone calcium (Ca-) salts. Of the 13 cholesterol-rich stones, nine stones had peripheral concentric layers of Ca-carbonate, whereas 2 stones had peripheral layers of Ca-phosphate. No Ca-salts were detected from 2 cholesterol-rich stones. The 2 stones containing Ca-phosphate had no Ca-salt cores, whereas the stones containing Ca-carbonate were separated into 3 different types: two stones with a Ca-carbonate core, four stones with several Ca-bilirubinate cores of glass-like structure, and 3 stones lacking Ca-salt cores. A closer view of the Ca-salt layers, which may be occasionally coexistent with Ca-bilirubinate, mainly showed either laminate deposits or numerous globules with a few laminae. Of the 2 cholesterol-poor stones, one had dispersed particles mainly of Ca-phosphate, and the other had loosely dispersed particles with small amounts of Ca-phosphate, bilirubinate, and/or palmitate. Some relationship between the size and Ca-salt species of these gallstones was suggested. Gallstones collected from the same individual showed a considerable heterogeneity of Ca-salts.

An ultrastructural evaluation of the effects of cysteine-proteinase inhibitors on osteoclastic resorptive functions.

This study was designed to evaluate the effects of specific and potent cathepsin inhibitors on osteoclastic resorptive functions in vitro by means of a novel ultrastructural assay system. Mouse bone marrow cell-derived osteoclasts were suspended on dentine slices and cultured for 48 hours in the presence of either E-64 (a generalized cysteine proteinase inhibitor) or Z-Phe-Phe-CHN2 (a selective cathepsin L inhibitor). After the removal of cultured osteoclasts, co-cultured dentine slices were examined using electron microscopy: backscattered (BSEM), scanning (SEM), and atomic force (AFM). In morphometric analyses of BSEM images, there were no significant differences in the areas of demineralized dentine surfaces between control and inhibitor-treated groups, suggesting that cathepsin inhibitors had no effect on dentine demineralization by cultured osteoclasts. However, in SEM and AFM observations, both inhibitors remarkably reduced to the same extent, the formation of deep resorption lacunae on dentine slices that had resulted from degradation of matrix collagen. In addition, Z-Phe-Phe-CHN2 treatment produced deeper, ring-like grooves with little collagen exposure in shallow resorption lacunae. These results strongly suggest that (1) cathepsins released by osteoclasts are involved in the formation of deep resorption lacunae, and (2) cathepsin L plays a key role in bone resorption.

Scanning electron microscopy and electron probe microanalysis studies of human pineal concretions.

The calcareous concretions of human pineal bodies were investigated with scanning electron microscopy and electron probe microanalysis. The initial concretions measuring 5-7 microns in diameter may have started at the calcified pinealocytes. They grew appositionally forming concentric laminations, and then the simple calcospherulites over 20 microns occasionally aggregated with each other. Some of them became numerous spherulite-aggregated concretions. Others individually grew with scallop-shaped concentric laminations at intervals of 0.05-1 microns and became lobated calcospherulites up to 0.5 mm. The concretions over 0.5 mm were formed by their attachments. The major elements were Ca and P, while traces of S, Mg, and Na were detected. In the calcification and crystallization values, the center of the concretions over 50 microns was significantly higher than the periphery, while there were no differences among the centers and also among the peripheries. The Ca and P amounts in the center were 30.8% and 14.2% by weight and the Ca/P molar ratio was 1.68; thereby the sand-grain-shaped crystals may be nearly hydroxyapatite, as reported previously.

Self-etching dentin primers containing phenyl-P.

The dentin bonding efficacies of two commercial dentin bonding systems and experimental self-etching dentin primers composed of methacryloxyethyl hydrogen phenyl phosphate (Phenyl-P) and either hydroxyethyl methacrylate (HEMA) or glyceryl methacrylate (GM,2,3-dihydroxypropyl methacrylate) were examined. The wall-to-wall polymerization contraction gap width of a commercial light-activated resin composite in a cylindrical dentin cavity and the tensile bond strength to a flat dentin surface were measured. Changes in dentin hardness were determined by Micro Vickers Hardness measurement, and an SEM observation was performed after priming. Formation of a contraction gap was completely prevented by the application of Phenyl-P diluted in HEMA or GM solution combined with a commercial dentin bonding agent, although gap formation was evident in nearly half of the specimens with both commercial dentin bonding systems. The mean tensile bond strengths of the tested groups varied from 16.3 to 20.7 MPa, and there were no significant differences between groups. Based on the measurement of Micro Vickers Hardness and SEM observation after priming, a slight reduction in dentin hardness was observed. However, this reduction in dentin hardness due to self-etching priming did not significantly correlate with either contraction gap width or tensile bond strength.

Scanning electron microscopy and energy-dispersive X-ray microanalysis studies of several human calculi containing calcium phosphate crystals.

Human calcium phosphate calculi: two sialoliths, a urolith, a rhinolith, and a tonsillolith were investigated by scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDX). The sialoliths and urolith had appositional shells with thick cortices, respectively, around several nuclei composed of calcospherulites and a rubber-film fragment. The rhinolith had a thin cortex with appositional laminations around a glomerulus-like mass of calcified cotton-like strings. The tonsillolith had a rough cortex with appositional laminations. Its porous interior was composed of numerous calcified conglomerates with microorganisms and calcified masses with fine appositional laminations around the conglomerates. The major crystals were identified as biological apatites (AP) with a sand-grain rather than a needle-like shape, and plate-shaped octacalcium phosphate (OCP). The AP deposits of the rhinolith probably were associated with magnesium (Mg) phosphates or contained Mg. No OCP was found in the rhinolith. The AP deposits were mainly formed by extracellular calcification. Hexahedral crystals, identified as Mg-containing whitlockite (WH), were precipitated in the internal spaces of the AP and OCP deposits. The rhinolith nucleus consisted of WH crystal deposits only.

Effect of delayed light curing of a resin composite on marginal integrity in cylindrical dentine cavities.

The effect of delayed light curing of resin composite on marginal adaptation has been examined by measuring the wall-to-wall polymerization contraction gap when using a commercial resin composite together with experimental dentine bonding systems to restore cylindrical preparations in dentine. Morphological changes in dentine during dentine bonding procedures were observed using a scanning electron microscope. In a previous report, the contraction gap width for a resin composite increased when irradiation of the resin system was delayed, despite the use of a dentine bonding system considered to be 'contraction' gap free. Such deterioration in marginal adaptation was minimized by use of an experimental dentine primer, 40% erythritol methacrylate aqueous solution (EM), followed by the use of a commercial dual- or autocured dentine bonding agent. Under scanning electron microscopy, the dentine surface microstructure became unclear after EM priming, and a polymer film was detected after polymerization of the dual-cured dentine bonding agent. The hydrogelled primer and the formation of a polymer network on the dentine surface may prevent the flow of fluid from the pulp through the dentine tubules, and maintain marginal integrity if there is delay in light curing of light-activated resin composite systems.

Measurement of Howship's resorption lacunae by a scanning probe microscope system.

We have developed a novel ultrastructural assay system for osteoclastic resorptive function. After osteoclasts had been co-cultured on dentine slices for 48 hr, the slices were fixed with glutaraldehyde and examined by means of backscattered electron, scanning electron, and scanning probe microscopies. Backscattered electron images showed areas of low mineralization on dentine surfaces, which, by superimposition of concave-convex images, corresponded to resorption lacunae. The measurement of such resorption lacunae by scanning probe microscopy revealed 3-dimensional topography and their exact depths and volumes. Analysis based on this system provides reliable qualitative and quantitative assessment of osteoclastic resorption.

Cytochalasin D reduces osteoclastic bone resorption by inhibiting development of ruffled border-clear zone complex.

The osteoclastic cytoskeleton has been demonstrated to be composed of microfilaments. Osteoclastic multinucleated cells were suspended on dentine slices and cultured for 24 hours in the presence or absence of cytochalasin D (CD), a specific and potent inhibitor of actin filament elongation to determine the role of this cytoskeleton. Cultured cells and co-cultured dentine slices were examined ultrastructurally. Unlike those in control cultures without CD, osteoclasts in CD-treated cultures became spherical in shape and lacked microvilli on their basolateral cell surfaces. Most importantly, CD treatment induced a complete disappearance of the ruffled border-clear zone complexes in osteoclasts, which resulted in loss of osteoclast-cytoplasmic polarity. Morphometric analysis of backscattered electron micrographs of co-cultured dentine slices revealed that CD treatment strongly inhibited the formation of resorption lacunae in a dose-dependent manner. These results suggest that the cytoarchitecture, as well as the bone-resorbing function, of the osteoclast is highly regulated by the F-actin-containing microfilamentous cytoskeleton in the ruffled border-clear zone complex.

Application of the environmental SEM in human dentin bleached with hydrogen peroxide in vitro.

The environmental SEM (E-SEM) can be used unfixed biological samples under a low vacuum and wet condition. In this study, the fractured dentin of unfixed human teeth was treated with a 30% hydrogen peroxide solution (H2O2) for the examination of tooth-bleaching prior to the E-SEM and a conventional SEM. The peritubular matrix (PM) always showed a few cracks along the long axis of a dentinal tubule, and the ends of fine fibrils rose to the smoothly changed surface of the intertubular matrix (IM). The E-SEM with non-fixation and the conventional SEM following fixation indicated that the hydrogen peroxide solution easily permeated the PM and dissolved the non-fibrillar substance including the cracks of the PM by the constriction. In the IM, the solution may partially dissolve the organic parts within mineralized fibrils as well as non-fibrillar substance between the fibrils, although these remnants might precipitate again there.

Scanning electron microscopy and energy-dispersive X-ray microanalysis studies of early dental calculus on resin plates exposed to human oral cavities.

Dental calculus formed after 10 days on resin plates, applied to the lingual sides of the mandibular gingival regions in eight human subjects, was investigated by means of scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDX). The mineral deposits were mainly divided into three types: A, B, and C. The type A deposits showing an average Ca/P molar ratio of 1.42 were densely packed with fine needle-shaped crystals formed by the intra- and extra-cellular calcification. The type A deposits, probably composed of Ca-deficient apatites and the transitional forms between apatite and octacalcium phosphate (OCP), were observed in all subjects. The type B deposits showing an average Ca/P molar ratio of 0.96 were aggregated with polygonal column, triangular plate-shaped, and rhombohedral crystals. These crystals identified as brushite (CaHPO4-2H2O:dicalcium phosphate dihydrate: DCPD) were found in four subjects. Platelet-shaped crystals of the type C deposits were observed in three subjects. Their Ca/P molar ratio of 1.26 and the crystal shape were similar to those of OCP. Whitlockite crystals were not found although Mg-containing hexagonal disk-like crystals were observed in two subjects.

Hexahedrally based crystals in human tooth enamel.

Mg-containing calcium phosphate crystals including pseudocuboidal, rhombohedral shapes and groupings of quadrangular blades cubically arranged were found in human tooth enamel by scanning electron microscopy and by electron probe microanalysis. In caries-free old enamel, these hexahedrally based crystals measuring 0.5-2.5 microns in length were observed in some crevices of tufts and lamellae. The crystals were rarely seen in the inner crevices of caries-free exfoliated deciduous enamel and none could be seen in sound young enamel. In brown-coloured old enamel possessing arrested caries with lamellae, some of the lamellae contained crystals measuring 0.1-1.5 mu in length adjacent to half-dissolved prisms. These crystals, identified as Mg-containing whitlockite, will grow during a long period after eruption of the tooth or during the enamel caries process.

Physico-chemical and morphological studies of horse dentin.

The well-developed thick peritubular matrix (PM) in the middle layer of horse dentin was physico-chemically and also morphologically compared with the intertubular matrix (IM) and the enamel using several microscopes. In scanning laser microscopy, the PM showed a higher laser-beam reflection than the IM, and the contrast image was similar to that of the backscattered electrons by SEM, but to be lower than that of X-ray opacity in microradiography. By SEM-EDX, Ca, P, and Mg were detected from the PM and IM. The mineral content of the PM composed of 15-20 nm sandygrain-shaped crystallites under SEM was significantly higher than that of the IM, but lower than the enamel. The PM/IM in each Ca and P content by weight showed a low average of 1.11, which corresponded to that of their specific gravities previously reported. In Vickers hardness tests, the PM was significantly harder than the IM but softer than the enamel. The PM/IM in the microhardness number showed a high average of 1.85.