Deficiency of chemokine receptor CCR1 causes osteopenia due to impaired functions of osteoclasts and osteoblasts.

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1(-/-) mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1(-/-) mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1(-/-) mice. Osteoclastogenesis induced from cultured Ccr1(-/-) bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1(-/-) osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1(-/-) mice. The co-culture of wild-type osteoclast precursors with Ccr1(-/-) osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.

In the absence of a basal lamina, ameloblasts absorb enamel in a serumless and chemically defined organ culture system.

OBJECTIVES: Tooth organ development was examined in a serumless, chemically defined organ culture system to determine whether morphological and functional development was identical to that in in vivo and serum-supplemented organ cultures. METHODS: Mouse mandibular first molar tooth organs at 16 days of gestation were cultured for up to 28 days in a Tronwell culture system using a serum-supplemented or serumless, chemically defined medium. After culture, specimens were processed for assessing tooth development using ultrastructural, immunohistochemical, and mRNA expression analyses. RESULTS: In serum-supplemented conditions, inner enamel epithelial cells differentiated into secretory-stage ameloblasts, which formed enamel and reached the maturation stage after 14 and 21 days of culture, respectively. Ameloblasts deposited a basal lamina on immature enamel. Conversely, in serumless conditions, ameloblasts formed enamel on mineralized dentin after 21 days. Moreover, maturation-stage ameloblasts did not form basal lamina and directly absorbed mineralized enamel after 28 days of culture. RT-PCR analysis indicated that tooth organs, cultured in serumless conditions for 28 days, had significantly reduced expression levels of ODAM, amelotin, and laminin-322. CONCLUSIONS: These results indicate that several differences were detected compared to the development in serum-supplemented conditions, such as delayed enamel and dentin formation and the failure of maturation-stage ameloblasts to form basal laminae. Therefore, our results suggest that some factors might be required for the steady formation of mineralized dentin, enamel, and a basal lamina. Additionally, our results indicate that a basal lamina is necessary for enamel maturation.

Trabecular structure and composition analysis of human autogenous bone donor sites using micro-computed tomography.

OBJECTIVES: To evaluate the bone microstructure of autogenous graft bone in elderly people (mean age, 66 years), we compared the bone volume/total volume and bone mineral density of four donor sites that are commonly harvested for maxillofacial surgery and dental implant treatments, using X-ray micro-computed tomography. METHODS: Eighteen Japanese cadavers were included in this study. Overall, 66 harvested bones (mandibular symphysis, mandibular ramus, ilium, and tibia) were studied. Micro-computed tomography scans of four sites were performed to analyze the trabecular structures, bone mineral density, and bone volume/total volume in these bones. RESULTS: The mandibular symphysis bones showed the highest bone volume/total volume and bone mineral density at the four sites. There was a significant difference in the bone volume/total volume between the mandibular symphysis and tibia groups. There was also a significant difference in bone mineral density between the mandibular symphysis group and the ilium and tibia groups. In the three-dimensional observations, the structures of the mandibular trabecular were plate-type. The structures of the tibial bone were mixtures of plate- and rod-types. In the ilium, most trabecula were rod-shaped. CONCLUSIONS: Mandibular symphysis and ramus had a higher bone volume/total volume and bone mineral density of the four sites and did not show regressive changes in our findings. Mandibular bone is the most suitable source of autogenous graft bone material because of its superior bone quality and quantity.

Effect of nitrogen-containing bisphosphonates on osteoclasts and osteoclastogenesis: an ultrastructural study.

We have previously indicated that a single injection of alendronate, one of the nitrogen-containing bisphosphonates (NBPs), affects murine hematopoietic processes, such as the shift of erythropoiesis from bone marrow (BM) to spleen, disappearance of BM-resident macrophages, the increase of granulopoiesis in BM and an increase in the number of osteoclasts. NBPs induce apoptosis and the formation of giant osteoclasts in vitro and/or in patients undergoing long-term NBP treatment. Therefore, the time-kinetic effect of NBPs on osteoclasts needs to be clarified. In this study, we examined the effect of alendronate on mouse osteoclasts and osteoclastogenesis. One day after the treatment, osteoclasts lost the clear zone and ruffled borders, and the cell size decreased. After 2 days, the cytoplasm of osteoclasts became electron dense and the nuclei became pyknotic. Some of the cells had fragmented nuclei. After 4 days, osteoclasts had euchromatic nuclei attached to the bone surface. Osteoclasts had no clear zones or ruffled borders. After 7 days, osteoclasts formed giant osteoclasts via the fusion of multinuclear and mononuclear osteoclasts. These results indicate that NBPs affect osteoclasts and osteoclastogenesis via two different mechanisms.

Resorption analysis of deproteinized cancellous bovine bone.

Commercially available deproteinized cancellous bovine bone (DPBB) has been indicated as non-absorbable bone filling materials. Stoichiometric hydroxyapatite (HA) was not resorbed by osteoclasts while calcium-deficient and carbonate-rich apatite converted from octacalcium (OCP hydrolysate) was resorbed by osteoclasts. We analyzed the chemical composition of DPBB and compared the tissue reactions around two materials after implanting into mouse bone marrow. X-ray diffraction analysis and Fourier transform infrared spectroscopy showed that DPBB was a carbonate-rich apatite. Micro-CT analysis indicated the massive bone formation on both materials at 2 weeks, then gradually resorbed. At 12 weeks, osteoclasts were directly attached to both materials. The ultrastructure of ruffled borders on DPBB was identical to osteoclasts resorbing normal bone while ruffled border on OCP hydrolysate showed irregular shape. These results indicated that DPBB was the absorbed material and that the structure of ruffled border of osteoclasts might be regulated by the size or orientation of HA.

Fine structure and mineral components of primary calculi in some human prostates.

The fine structure of prostatic calculi has not been elucidated yet, although the chemical components were reported in detail. We studied the primary or endogenous calculi removed from eight human prostates by secondary scanning electron microscopy, backscattered electron imaging, energy-dispersive X-ray microanalysis and X-ray diffraction. The primary calculi containing Mg, Zn and S, besides Ca and P were basically classified into four stone groups (I-IV) by fine structure and mineral components. Stone I had the core deposits of calcospherites showing concentric rings and the laminated deposits concentrically around the core. Their deposits were identified as apatite. Stone II was occupied with the calcospherite deposits of apatite although the stone growth showed a rough concentric formation. Stone III contained the core of calcospherites and concentric laminated structures, similar to a smaller type of group I, whereas the wider peripheral region was deposited with needle-like structures, identified as calcium oxalates. Stone IV had the core deposits containing small hexahedral structures, identified as whitlockite, which were surrounded with several incompletely concentric laminated bands of apatite. Whitlockite crystals were also found between the fused large calculi. The initial and formative calculi were basically observed as the deposition of mineralizing spherical structures suggesting variously sized corpora amylaceous bodies. Thus, the primary prostatic calculi of stones I-III will begin from the mineralization of amylaceous bodies as a core, while the organic substances, which form stone IV, might be derived from the simple precipitation of prostatic secretion.

Cellular mechanism of inhibition of osteoclastic resorption of bone and calcified cartilage by long-term pamidronate administration in ovariectomized mature rats.

We examined the effects of long-term bisphosphonate (BP, pamidronate) administration at a therapeutic dose (1.5 mg/kg/day) on the distribution, structure, and vacuolar-type H(+)-ATPase expression of osteoclasts, and the resulting trabecular bone volume and structure in ovariectomized (OVX) mature rats. Six-month-old female rats were allocated to sham-operated control, untreated-OVX, and BP-administered OVX groups. Postoperatively, BP was administered intraperitoneally once a day to OVX rats for up to 30 days. On postoperative days 14, 30, and 60, all of the rats were killed and the distal metaphyseal area of the dissected humeri was examined. Quantitative backscattered-electron image analysis revealed that the trabecular bone volume/unit medullary area in untreated OVX rats was significantly (P < 0.05) lower than that in sham-operated controls at 30 and 60 days postoperation. BP administration significantly (P < 0.05) increased trabecular bone volume at 14, 30, and 60 days postoperation in BP-administered OVX rats compared to both sham-operated and untreated OVX rats. Compared to untreated OVX rats, the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts along the bone trabeculae in BP-administered OVX rats was not significantly decreased on days 14 and 30, but was significantly decreased on day 60. Ultrastructurally, BP administration caused the disappearance of both the ruffled border (RB) and the clear zone (CZ) structures, and decreased the expression of vacuolar-type H(+)-ATPase in most osteoclasts, but did not significantly induce apoptosis of osteoclasts detected by the terminal dUTP nick end-labeling (TUNEL) method. Our results suggest that long-term BP administration significantly reduces bone and calcified cartilage resorption through impairment of the structure and bone-resorbing function of osteoclasts, and thereby effectively maintains trabecular bone volume and structure in ovariectomy-induced acute estrogen deficiency in mature rats.

Effects of surface roughness and dimorphism on the adhesion of Candida albicans to the surface of resins: scanning electron microscope analyses of mode and number of adhesions.

AIM: Candida albicans is a common oral fungus but can cause serious conditions such as Candida stomatitis. We investigated C. albicans adhesion to the surface of denture-base resins at two growth phases. METHODS: Fungal suspensions of logarithmic (9 h) and stationary phase (24 h) C. albicans (JCM2085) were used. Scanning electron microscopy (SEM) confirmed that yeast and mycelial forms were predominant in 9-h and 24-h cultures, respectively. Resin strips were polished to three surface roughness levels (Ra 3.2 µm, Ra 0.48 µm and Ra 0.06 µm) and were then immersed in C. albicans suspensions for both phases. The SEM images were taken at five sites on each strip. RESULTS: Adhesion of mycelial-form C. albicans on rough surfaces (Ra = 3.2) was 2.2 times higher than on smooth surfaces (Ra = 0.06; 7030 vs 3580 adhesions/mm(2), P < 0.01). The hyphae of these mycelial forms fully penetrated the surface cracks. Fewer adhesions occurred for yeast-form C. albicans, regardless of surface type (440-620 adhesions/mm(2), P = n.s.). CONCLUSION: Adhesion of yeast-form C. albicans was indifferent to surface roughness. In contrast, mycelial adhesion increased with surface roughness of the resin because mycelia infiltrated the minute protuberances on rough surfaces.

Honeycomb form ß-tricalcium phosphate induces osteogenesis by geometrical property with BMSC.

To establish an effective method for bone augmentation, we introduced a new honeycomb-like ß-tricalcium phosphate (H-ß-TCP) with BMP-2 as a scaffold, whose unique geometrical properties induce osteoblastic differentiation of autologous bone marrow mesenchymal stem cells (BMSCs). A total of six beagle dogs from 6 to 7 years old were used for this study. BMSCs were cultured with autologous serum and BMP-2 on H-ß-TCP. Differentiation to osteoblasts was demonstrated in vitro and exo vivo. Scanning electron microscopy revealed formation and calcification of a matrix-like structure within the H-ß-TCP tunnels in BMSC culture. Moreover, treatment of BMP-2 promoted osteoblastic differentiation of BMSCs in H-ß-TCP in a diffusion chamber. These results indicated that H-ß-TCP may be a useful tool for construction of functional artificial bone.