Tartrate-resistant Acid Phosphatase Histochemistry Research Articles

Effect of Hydroxyapatite/β-Tricalcium Phosphate on Osseointegration after Implantation into Mouse Maxilla

In our previous study we established an animal model for immediately placed implants using mice and clarified that there were no significant differences in the chronological healing process at the bone-implant interface between immediately and delayed placed implants blasted with hydroxyapatite (HA)/β-tricalcium phosphate (β-TCP) (ratio 1:4). This study aimed to analyze the effects of HA/β-TCP on osseointegration at the bone-implant interface after immediately placed implants in the maxillae of 4-week-old mice. Right maxillary first molars were extracted and cavities were prepared with a drill and titanium implants, blasted with or without HA/β-TCP, were placed. The fixation was followed-up at 1, 5, 7, 14, and 28 days after implantation, and the decalcified samples were embedded in paraffin and prepared sections were processed for immunohistochemistry using anti-osteopontin (OPN) and Ki67 antibodies, and tartrate-resistant acid phosphatase histochemistry. The undecalcified sample elements were quantitatively analyzed by an electron probe microanalyzer. Bone formation occurred on the preexisting bone surface (indirect osteogenesis) and on the implant surface (direct osteogenesis), indicating that osseointegration was achieved until 4 weeks post-operation in both of the groups. In the non-blasted group, the OPN immunoreactivity at the bone-implant interface was significantly decreased compared with the blasted group at week 2 and 4, as well as the rate of direct osteogenesis at week 4. These results suggest that the lack of HA/β-TCP on the implant surface affects the OPN immunoreactivity on the bone-implant interface, resulting in decreased direct osteogenesis following immediately placed titanium implants.

Osteopontin on the Dental Implant Surface Promotes Direct Osteogenesis in Osseointegration.

After dental implantation, osteopontin (OPN) is deposited on the hydroxyapatite (HA) blasted implant surface followed by direct osteogenesis, which is significantly disturbed in Opn-knockout (KO) mice. However, whether applying OPN on the implant surface promotes direct osteogenesis remains unclarified. This study analyzed the effects of various OPN modified protein/peptides coatings on the healing patterns of the bone-implant interface after immediately placed implantation in the maxilla of four-week-old Opn-KO and wild-type (WT) mice (n = 96). The decalcified samples were processed for immunohistochemistry for OPN and Ki67 and tartrate-resistant acid phosphatase histochemistry. In the WT mice, the proliferative activity in the HA binding peptide-OPN mimic peptide fusion coated group was significantly higher than that in the control group from day 3 to week 1, and the rates of OPN deposition and direct osteogenesis around the implant surface significantly increased in the recombinant-mouse-OPN (rOPN) group compared to the Gly-Arg-Gly-Asp-Ser peptide group in week 2. The rOPN group achieved the same rates of direct osteogenesis and osseointegration as those in the control group in a half period (week 2). None of the implant surfaces could rescue the direct osteogenesis in the healing process in the Opn-KO mice. These results suggest that the rOPN coated implant enhances direct osteogenesis during osseointegration following implantation.

Calcification and resorption of mouse Meckel's cartilage analyzed by von Kossa and tartrate-resistant acid phosphatase histochemistry and scanning electron microscopy/energy-dispersive X-ray spectrometry.

Meckel's cartilage is essential for the normal development of the mandible. The fate of the intermediate portion of Meckel's cartilage is unique as most of it disappears soon after birth except for the part that forms the sphenomandibular ligament. The mechanism of the disappearance of Meckel's cartilage is unknown; therefore, this study was designed to investigate the process of Meckel's cartilage degradation, focusing on cartilage matrix calcification and the appearance of chondroclasts. Developing mouse mandibles at embryonic days 15, 16, 17, and 18, and postnatal day 2 were processed for whole-mount staining with alcian blue and alizarin red. The mandibles on embryonic days 15, 16, 17, and 18 were fixed and embedded in paraffin. Adjacent sections were processed for von Kossa and tartrate-resistant acid phosphatase (TRAP) histochemistry and scanning electron microscopy/energy-dispersive X-ray spectrometry (SEM/EDS). Calcification and the element concentrations of calcium, phosphorus, and carbon were examined with von Kossa histochemistry and SEM/EDS. The involvement of chondroclasts was investigated using TRAP histochemistry. The results demonstrated that the intermediate portion of Meckel's cartilage is resorbed by chondroclasts after chondrocyte hypertrophy and cartilage matrix calcification and that the mineral concentration of calcified Meckel's cartilage is comparable to that of the surrounding bone. This study contributes to the understanding of the mechanism of Meckel's cartilage resorption and provides useful insights into the development of the mandible.

Differences in Healing Patterns of the Bone-Implant Interface between Immediately and Delayed-Placed Titanium Implants in Mouse Maxillae.

There are no available data on the healing process at the bone-implant interface after immediate implant placement. This study aimed to establish an animal experimental model of titanium implants placed in mouse maxillae and compare the healing pattern of the bone-implant interface after immediate implant placement with that after delayed implant placement. Maxillary first molars (M1) from 4-week-old mice were extracted and replaced with the implant following drilling (immediate-placement group). In contrast, M1 from 2-week-old mice were extracted, followed by drilling and implantation after 4 weeks (delayed-placement group). The decalcified samples at 0-28 days after implantation were processed by immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and tartrate-resistant acid phosphatase histochemistry. The elements and bone volume of undecalcified samples were quantitatively analyzed by an electron probe microanalyzer. Osseointegration was completed by 28 days after the procedure in both groups. There were no differences in contact area, bone loss at the cervical area, or rate of calcification at the bone-implant interface between the two groups. This study found no significant differences in the chronological healing process at the bone-implant interface between the two groups at the cellular level.

Prednisolone induces osteoporosis-like phenotype in regenerating zebrafish scales

We demonstrate that glucocorticoids induce an osteoporotic phenotype in regenerating scales of zebrafish. Exposure to prednisolone results in altered mineral content, enhanced matrix breakdown, and an osteoporotic gene-expression profile in osteoblasts and osteoclasts. This highlights that the zebrafish scale provides a powerful tool for preclinical osteoporosis research. This study aims to evaluate whether glucocorticoid (prednisolone) treatment of zebrafish induces an osteoporotic phenotype in regenerating scales. Scales, a readily accessible dermal bone tissue, may provide a tool to study direct osteogenesis and its disturbance by glucocorticoids. In adult zebrafish, treated with 25 μM prednisolone phosphate via the water, scales were removed and allowed to regenerate. During regeneration scale morphology and the molar calcium/phosphorus ratio in scales were assessed and osteoblast and osteoclast activities were monitored by time profiling of cell-specific genes; mineralization was visualized by Von Kossa staining, osteoclast activity by tartrate-resistant acid phosphatase histochemistry. Prednisolone (compared to controls) enhances osteoclast activity and matrix resorption and slows down the build up of the calcium/phosphorus molar ratio indicative of altered crystal maturation. Prednisolone treatment further impedes regeneration through a shift in the time profiles of osteoblast and osteoclast genes that commensurates with an osteoporosis-like imbalance in bone formation. A glucocorticoid-induced osteoporosis phenotype as seen in mammals was induced in regenerating scalar bone of zebrafish treated with prednisolone. An unsurpassed convenience and low cost then make the zebrafish scale a superior model for preclinical studies in osteoporosis research.

Histological and elemental analyses of impaired bone mineralization in klotho‐deficient mice

The klotho gene-deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4- and 5-week-old klotho-deficient mice (klotho(-/-)mice) and their wild-type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin-embedded specimens were used for detecting mineralization - according to von Kossa's staining method - and for elemental mapping by electron probe micro-analyzer, respectively. Alkaline phosphatase-positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho(-/-)metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase-positive osteoclasts. The wild-type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well-mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho(-/-)mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non-mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro-analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho(-/-)mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less-developed cell organelles in the klotho(-/-)mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.

Root Resorption on Torqued Human Premolars Shown by Tartrate-Resistant Acid Phosphatase Histochemistry and Transmission Electron Microscopy

To identify clastic cells on the root surfaces of torqued human premolars. A continuous force of 600 cNmm was applied to upper first premolars in patients 13-16 years of age by using a precise biomechanical model with superelastic wires (NiTi-SE). The 28 teeth in 14 patients were divided into five groups (control [nonmoved], and moved for either 1, 2, 3, or 4 weeks) and processed for tartrate-resistant acid phosphatase (TRAP) histochemistry and transmission electron microscopy. Mononuclear TRAP-positive cells appeared at 2 weeks, where as large multinucleated TRAP-positive cells were numerous at 3 and 4 weeks. Ultrastructural examination revealed many clastic cells in contact with resorption lacunae. In addition, some cementoblast-like cells appeared secreting new cementum over previously resorbed lacunae. In general, resorption lacunae and the number of clastic cells, which increased with the duration of the applied force, were found on the cementum surface at the pressure areas. Some signs of cementum repair were also noticed, even with the maintenance of the level of the force.