Vitamin K2, a gamma-carboxylating factor of gla-proteins, normalizes the bone crystal nucleation impaired by Mg-insufficiency.

It has been reported that the Mg-insufficient bone is fragile upon mechanical loading, despite its high bone mineral density, while vitamin K2 (MK-4: menatetrenone) improved the mechanical strength of Mg-insufficient bone. Therefore, we aimed to elucidate the ultrastructural properties of bone in rats with dietary Mg insufficiency with and without MK-4 supplementation. Morphological examinations including histochemistry, transmission electron microscopy, electron probe microanalysis (EPMA) and X-ray diffraction were conducted on the femora and tibiae of 4-week-old Wistar male rats fed with 1) a normal diet (control group, 0.09% Mg), 2) a Mg-insufficient diet (low Mg group, 0.006% Mg), or 3) a Mg-insufficient diet supplemented with MK-4 (MK-4 group, 0.006% Mg, 0.03% MK-4). MK-4 appeared to inhibit the osteoclastic bone resorption that is stimulated by Mg insufficiency. EPMA analysis, however, revealed an increased concentration of Ca paralleling Mg reduction in the low Mg group. Assessment by X-ray diffraction revealed an abundance of a particular synthetic form of hydroxyapatite in the low Mg group, while control bones featured a variety of mineralized crystals. In addition, Mg-deficient bones featured larger mineral crystals, i.e., crystal overgrowth. This crystalline aberration in Mg-insufficient bones induced collagen fibrils to mineralize easily, even in the absence of mineralized nodules, which therefore led to an early collapse of the fibrils. MK-4 prevented premature collagen mineralization by normalizing the association of collagen fibrils with mineralized nodules. Thus, MK-4 appears to rescue the impaired collagen mineralization caused by Mg insufficiency by promoting a re-association of the process of collagen mineralization with mineralized nodules.

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.

Reduced osteoblastic population and defective mineralization in osteopetrotic (op/op) mice.

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization.

Histochemical evidences on the chronological alterations of the hypertrophic zone of mandibular condylar cartilage.

The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the "articular zone," "articular layer," and "resting zone" from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles.

Postnatal development of excitation propagation in the trigeminal subnucleus caudalis evoked by afferent stimulation in mice.

The postnatal development of nociceptive afferent activity expansion and its modulation features were examined in mice using an optical imaging technique. Developing mice (1-2 weeks old (N1-2 w), 3-4 weeks old (N3-4 w), 5-6 weeks old (N5-6 w) and 7-8 weeks old (N7-8 w)) and neonatally capsaicin-treated mice were used. The propagation of neuronal excitation was measured by changes in fluorescent intensity in horizontal brain stem slices evoked by electrical stimulation to the trigeminal spinal tract. A single-pulse stimulation evoked excitation propagation in the trigeminal caudalis (Vc). The propagation area was larger in N1-2 w than in N7-8 w, and no differences were observed between capsaicin-treated and naive mice in the same age groups. Repetitive stimulation (100 Hz, 30 pulses) elicited long-lasting and widespread excitation propagation. The excitation propagation area was significantly larger in N7-8 w than in N1-2 w, N3-4 w and N5-6 w. This propagation was suppressed by 5 microM L-703.606, an NK1-receptor antagonist, suggesting that the repetitive stimulation-elicited excitation may require substance-P releases. Morphological observations demonstrated that the neural network in the Vc had grown by postnatal week 5. These results suggest that nociceptive afferent activity co-operatively matures with development of the network structure in the Vc, and that a mechanism for prolonged increase in central excitability is established during a later postnatal period.

Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis.

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.

Repetitive afferent stimulation propagates excitation in the trigeminal caudalis.

A high-speed optical imaging technique was employed for visualizing neuronal excitation propagation elicited by afferent stimulation in the mouse trigeminal caudalis (Vc) to clarify the central nociceptive modulation mechanism. Membrane depolarization evoked by a single-pulse stimulation to the spinal trigeminal tract (Tr) was propagated rostrally to the Vc, which was suppressed by CNQX. This is consistent with our morphological observation that axons expand from the Tr into the Vc. A trained-pulse (tetanus) stimulation to the Tr evoked a broad, persistent excitation in the Vc, while MK-801 suppressed it. Neonatally capsaicin-treated mice maintained a single-pulse response but a lacked tetanus-evoked one. These indicated that prolonged depolarization elicited by repetitive stimulation is a prerequisite to C-fiber excitation for activating the NMDA receptors.

Pulpal regeneration after cavity preparation, with special reference to close spatio-relationships between odontoblasts and immunocompetent cells.

The regeneration process of the odontoblast cell layer incident to tooth injury, especially its relationship with immunocompetent cells in pulp healing, has not been fully understood. The purpose of the present study was to clarify this relationship between odontoblasts and immunocompetent cells in the process of pulp regeneration following cavity preparation in rat molars by immunocytochemistry for heat shock protein (Hsp) 25 as well as class II major histocompatibility complex (MHC) molecules. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin, whereas class II MHC-positive cells were predominantly located beneath the odontoblast cell layer. Cavity preparation caused the destruction of the odontoblast layer to form an edematous lesion and the shift of class II MHC-positive cells with the injured odontoblasts toward the pulp core at the affected site. Some damaged odontoblasts without apparent cytoplasmic processes, round in profile, retained the immunoreactivity for Hsp25, suggesting the survival of a part of the odontoblasts against artificial external stimuli. Twelve hours after cavity preparation, numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules. By postoperative 72 hours, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border, but the class II MHC-positive cells moved from the pulp-dentin border to the subodontoblastic layer. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts. The functional roles of Hsp 25-positive odontoblasts and immunocompetent cells such as class II MHC-positive cells in the process of pulp regeneration after cavity preparation are discussed in conjunction with our previous experimental data.