Yukio Iwayama

Sintered carbonate apatites as bioresorbable bone substitutes

The dissolution behavior of sintered carbonate apatite was investigated in a 10 mM/L acetic acid solution adjusted to pH 5.0 at 37 degrees C, and compared to that of sintered hydroxyapatite and bone apatite for the purpose of establishing some similarities between the physicochemical dissolution of apatite biomaterials in vitro and their ability to be resorbed by osteoclasts in vivo. Both the sintered carbonate apatite and the bone apatite dissolved to an appreciable extent. Their solution compositions changed in an almost identical manner until toward the end of the reaction. The solution compositions for sintered carbonate apatite at 30 s was comparable with that for sintered hydroxyapatite at 3.8 days with respect to the degree of supersaturation, indicating that the former specimen is much more soluble than the latter specimen. Osteoclasts which were obtained from the long bones of 1-day-old neonatal rabbits resorbed bone and sintered carbonate apatite, but not sintered hydroxyapatite. These findings suggest that sintered carbonate apatites, which have characteristics that can be favorably compared with those of bone, especially with respect to its reactivity to acid media, would be useful as bioresorbable bone substitutes.

Osteoclastic responses to various calcium phosphates in cell cultures.

Disks made of hydroxyapatite, beta-tricalcium phosphate, carbonate apatite, tetracalcium phosphate, alpha-tricalcium phosphate, dicalcium phosphate dihydrate, and octacalcium phosphate were incubated in osteoclastic cell cultures for 2 days. The first five salts were sintered and the last two were compressed before incubation. Osteoclasts resorbed only the sintered carbonate apatite disks. However, osteoclasts were able to resorb octacalcium phosphate disks that were preincubated for 1 day in medium without cells, indicating that surface conditioning was important for osteoclastic resorption of this calcium phosphate. Although resorption did not occur, medium calcium and phosphorus changed to an appreciable extent after a 2-day incubation of beta-tricalcium phosphate, tetracalcium phosphate, alpha-tricalcium phosphate, and dicalcium phosphate dihydrate. These changes in the medium calcium and phosphate concentrations could explain why osteoclasts appeared to have lost their activity on these calcium phosphate disks and were not capable of resorbing them. With hydroxyapatite disks no changes were observed in the medium calcium and phosphorus before and after incubation. Moreover, the osteoclasts appeared to be essentially the same as with the sintered carbonate apatite disks and with bone slices used as a control. Nevertheless, no pits or lacunae were observed on the hydroxyapatite disks, indicating that sintered carbonate apatite should be superior to sintered hydroxyapatite as a bioresorbable bone substitute.

Periodontal tissue regeneration using fibroblast growth factor -2:Randomized controlled phase II clinical trial

Background The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. Methodology/Principal Findings We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured ≥3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 µL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified. Conclusions Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis. Trial Registration ClinicalTrials.gov NCT00514657

Formation of apatite—collagen complexes

An apatite-collagen complex was prepared in calcium beta-glycerophosphate solutions at pH 9.0 and 37 degrees C with the purpose of developing new bone substitutes that more closely resemble bone than currently available materials. Reconstituted type I collagen as well as sheet collagen were crosslinked in the presence of alkaline phosphatase and egg-yolk phosvitin. The crosslinked collagens were immersed in daily-renewed calcium beta-glycerophosphate solutions for 2 and 4 weeks to induce the deposition of apatite on the collagen fibers. After 2 weeks of reaction, for example, apatites deposited approximately two times the crosslinked collagen in weight. With reconstituted collagen, the complex showed some elasticity but no apatite was visually observed to detach under deformation with fingers and forceps. The complex, moreover, did not disintegrate when immersed in saline or animal blood. Nevertheless, the complex resorbed with no evidence of cytotoxicity when implanted in muscle tissues. These findings suggest that the apatite-collagen complex prepared would be useful as bone substitutes, especially for periodontal osseous lesion repair and alveolar ridge augmentation.

Development of a new calcium phosphate cement that contains sodium calcium phosphate

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.

Use of glass slides coated with apatite-collagen complexes for measurement of osteoclastic resorption activity.

This study was designed to evaluate the use of apatite-collagen complexes (ACC) coated onto glass slides for measurement of osteoclastic resorption activity. ACC-coated glass slides were prepared by immersion in beta-glycerophosphate solution for 7-14 days after glass slides coated with type I collagen had been treated with alkaline phosphatase and phosvitin. Osteoclast-containing cell suspensions were prepared from the long bones of 1-day-old rabbits and were seeded in medium 199 (containing 10% FBS) onto ACC-coated glass slides. After allowing the cells to attach for 1.5 h, the glass slides were incubated for periods of up to 96 h. The cells were observed by scanning electron microscopy and cytochemically for tartarate resistant acid phosphatase (TRAP) activity. Some slides were treated with FITC-phalloidin and anti-type I collagen antibody. TRAP-positive multinucleated cells were located in transparent spaces on the glass slides. These spaces did not stain immunohistochemically with anti-type I collagen antibody. Podosome formation was observed in the multinucleated cells facing the edge of the transparent spaces. The scanning electron microscopy demonstrated well-spread large cells located on the flattened surface on apatite particles covering the glass surface. Our results suggest that osteoclasts could resorb the apatite particles and coated collagen on the glass slide. The resorption lacunae appeared as transparent spaces, and the cytoskeleton of resorbing osteoclasts was observed in these spaces. ACC-coated glass slides could be useful for investigating the function and metabolic activities of osteoclasts.

Tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) stimulate osteoclastic bone resorption

Abstract: As both tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-2 have been reported to inhibit bone resorption, we examined whether TIMP-1 or TIMP-2 in fetal calf serum (FCS), with which culture media were supplemented, affected osteoclastic bone resorption in vitro. Contrary to our expectation, almost complete suppression of osteoclastic bone resorption was observed when both TIMP-1 and TIMP-2 were removed from the FCS. Bone resorption was, however, almost fully restored by the addition of recombinant TIMPs. TIMPs stimulate bone resorption at significantly lower concentrations (∼ng/ml) than those (∼μg/ml) required to inhibit bone resorption. To understand the mechanism of TIMP-dependent bone resorption, we counted and compared the number of tartrate-resistant acid phosphatase-(TRAP-) positive and multinuclear cells in cultures containing either 10% FCS or TIMP-1-free and/or TIMP-2-free FCS. There was essentially no difference in number among these, suggesting that the TIMP role seems to be related to the functional expression of osteoclasts. Metallo-proteinase inhibitors, either BE16627B[l-N-(N-hydroxy-2-isobutylsuccinynamoyl)-seryl-l-valine] or R94138 {N-methyl-(3S)-2-[(2R)-2-hydroxycarbamoylmethylundecanoyl] hexahydropyridazine-3-carboxamide}, could not replace TIMPs, suggesting that the osteoclast-stimulating activity of TIMPs cannot be ascribed to merely their inhibitory effect on matrix metalloproteinases.

Lectin-binding glycoconjugates and immunoreactive proteoglycans in resorption pits freshly excavated by isolated rabbit osteoclasts

Staining with FITC-conjugated concanavalin A, wheatgerm agglutinin and peanut agglutinin demonstrated that abundant sugar residues are present in resorption pits produced in vitro and over the cell surface of rabbit osteoclasts resorbing bovine femoral bone slices. After chondroitinase ABC digestion the stubs of chondroitin 4-sulphate and dermatan sulphate could be detected in the resorption pits by monoclonal antibodies.

Effect of Bisphosphonate on Guided Tissue Regeneration.

木研究の目的は組織再生誘導洪 (GTR法) を用いた歯槽骨再生過程において骨代謝疾患治療薬であるビスフォスフォネートの歯槽骨再生に対する影響を検討することにある。ビーグル成犬10頭の下顎前臼歯部に2壁性の人工骨欠損を作製し, これに吸収性膜 (RESOLUT ®) を用いGTR法を行った。実験群5頭にはビスフォスフォネート (アレディア ®) (0.6mg/kg) を, 対照群5頭には生理食塩水を筋肉注射により投与した。術後11週目まで毎週血液採取と規格レントゲン写真を撮影した。採取した血液から血清を分離し, 骨形成マーカーとしてのオステオカルシン (OC) 量を測定した。規格レントゲン写真から画像解析を行い骨密度を測定した。その結果, 両群の血清中OC変動率は骨欠損作製前に比べ術後3日目で最大値を示し, その後徐々に減少していった。実験群のOC変動率は1～2週目まで対照群と比較して高い推移を示した。規格レントゲン写真の画像解析の結果, 実験群の骨密度は3～5週目において対照群と比較して高い値を示した。以上の結果より, ビスフォスフォネート投与はGTR法における歯槽骨再生を促進させることが示唆された。

Microassay for Sulfated Glycosaminoglycans by Alcian Blue Staining. Effects of Polyanionic Macromolecules on Staining in Untreated Biological Samples.

グリコサミノグリカンは酢酸セルロース膜を用いた電気泳動によって分離の後アルシアンブルーによって検出されてきた。著者らはすでにアルシアンブルーと陰イオンを結合させる方法を用いてDNA, ヒアルロン酸, 牛血清アルブミンなどの他の陰性荷電物質の妨害を受けないで硫酸化グリコサミノグリカンを直接微量分析する方法を報告した。本研究の目的は生体液に存在する他の陰性荷電高分子物質が本法による硫酸化グリコサミノグリカンの直接分析を妨害しないかどうか検討することである。2μlのサンプルを酢酸セルロースのペーパーストリップスにしみこませ, これをpH1.5に調製した0.05M塩化マグネシウム, 0.4Mグアニジン塩酸, 0.02M硫酸, 0.25%トライトンX-100を含む0.2%アルシアンブルー溶液で染色した。室温で乾燥後, 染色部分を2波長クロマトスキャナーで分析した。ヒト唾液, ヒト血清, ヒト膝関節液中の陰性荷電高分子物質と結合する染色剤の量は少なかった。精製した歯肉硫酸化グリコサミノグリカンについて電気泳動法と本法の結果を比較したところ, 本法で測定した硫酸化グリコサミノグリカン量は電気泳動法の結果の約53%であった。本測定法は技術的に簡単で, 要する時間は短くまた感度が高く, また他の陰性荷電物質が存在する生体液にも応用できるが, 検出率をさらに改良する必要がある。