Tatsushi Kawai

Osteoinductive activity of composites of bone morphogenetic protein and pure titanium

Titanium sponges were infused with bone morphogenetic protein (BMP-Ti), and the osteoinductivity of the resultant composite was measured. New bone formation occurred three weeks after implantation and was identified by soft x-ray analysis. Quantitative analysis showed no significant difference between BMP-Ti composites and control samples (BMP only). Consequently, pure titanium neither inhibited nor promoted BMP activity. Chondrocytes and new bone formation occurred in direct contact with the surfaces of the titanium. X-ray microanalysis demonstrated new bone formation inside the pores of the titanium sponges. The BMP-Ti composite has interesting properties as an osteoinductive implant and has potential practical clinical applications.

Identification of specific autoantigens in Sjögren's syndrome by SEREX.

We carried out SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using sera from patients with Sjögrens syndrome (SjS) and investigated the frequencies of autoantibodies against autoantigens identified by SEREX in the sera of healthy individuals (HI) and patients with SjS, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). IFI16 and two kelch‐like proteins, KLHL12 and KLHL7, were found to be novel autoantigens in SjS by SEREX. A markedly high frequency of anti‐IFI16 autoantibodies was observed in the sera of SjS (SjS, 70%; RA, 13%; SLE, 33%; HI, 0%). Interestingly, all serum samples from SjS demonstrated immunoreactivity against one or both of IFI16 and SS‐B/La. The presence of autoantibodies against KLHL12 and KLHL7 in the sera was significantly specific to SjS (23% and 17%, respectively), as they were not detected in RA, SLE or HI. Furthermore, we confirmed that transcripts of these autoantigens were expressed preferentially in the salivary glands and immuno‐privileged testes. Our results suggest these autoantigens may be useful as serological markers for the clinical diagnosis of SjS and may play a crucial role as organ‐specific autoantigens in the aetiopathogenesis of SjS. This study warranted clinical evaluations of autoantibodies against IFI16, KLHL12 and KLHL7 in combination with anti‐SS‐B/La autoantibodies.

Changes in Collagen Types During the Healing of Rabbit Tooth Extraction Wounds

Three α chains of type V collagen - α1(V), α2(V), and α3(V)- were initially demonstrated together with the expected collagen types I and III in the pepsin-soluble fraction of both normal mandibular bone and tooth extraction wound tissues of rabbits, as analyzed by sodium dodecyl sulfate-gel electrophoresis. The total collagen content of each extraction wound, as determined by the hydroxyproline assay, was observed to increase continuously from day 5 through day 17 and then leveled off or decreased. The ratio of type V to type I collagen was significantly higher in the initial stage of wound healing and decreased sharply down to the level of mandibular bone by day 5. The ratio of type III to type I collagen in the pepsin-soluble fraction increased and reached a maximum on day 5, whereas it was maximal on day 7 in the cyanogen bromide-soluble fraction, and thereafter decreased gradually in both fractions. The ratio for the pepsin-soluble fraction was, however, significantly higher than that for the cyanogen bromide-soluble fraction in the early stage of wound healing.

The effect of heat-treated human bone morphogenetic protein on clinical implantation.

For the clinical usage of human-derived bones, it is necessary to treat bones to reduce the risk of contamination by microorganisms. Bone morphogenetic protein is vulnerable to chemicals, but shows resistance to thermal heat to 70° C in a short time. In this experiment, crude human bone morphogenetic protein was extracted from heat-treated bones at 60° C for 10 hours and from nonheated bones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis for these specimens was done. Gelatin capsules containing 5 mg of crude human bone morphogenetic protein extracted from heated and nonheated bones were implanted into thigh muscle pouches of five mice. At 20 days after implantation, the heterotopic bone formation was compared by evaluating the radiographic and histologic analyses. The sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of the human bone morphogenetic proteins showed five main bands (16, 22, 28, 35, and 67 kDa) that were almost identical. Heterotopic bone formation observed on the radiograph was induced by crude human bone morphogenetic protein from heated bones in a manner similar to that used for nonheated bones. The results from this study show that heat-treated bone preserves osteoinduction.

Evaluation of heterotopic bone formation induced by squalane and bone morphogenetic protein composite

Bone morphogenetic protein is an important molecule whose bioactivity depends on the carrier. Squalane is used in the formulation of various kinds of cosmetics because it is easily emulsified and has the property of spreading well. Thus, squalane might be effective as a bone morphogenetic protein delivery system. As a test for this possibility, gelatin capsules containing squalane and bone morphogenetic protein (bovine derived partially purified) composite were implanted under the hindquarter perimuscular membrane of ddY mice. Control capsules containing only bone morphogenetic protein were used for controls. The implants were radiographically and histologically examined at 1 to 4 weeks after the operation. According to the radiographic analysis, squalane and bone morphogenetic protein composite and bone morphogenetic protein only control specimens formed widespread heterotopic bone tissues. The amount of heterotopic bone formation in the composite experimental specimens was approximately 40% greater than that in the controls. Histologic examination of experimental and control specimens revealed varying amounts of perichondral ossification by 2 weeks. By 3 and 4 weeks, the bone deposits were colonized by hematopoietic bone marrow. Squalane was effective for the slow local release of bone morphogenetic protein. Furthermore, the squalane and bone morphogenetic protein composite was a reliable osteoinductive biomaterial.

Methyl Methacrylate Activates the Gsta1 Promoter

Residual monomers in resin-based biomaterials cause cytotoxicity. We previously showed that methyl methacrylate (MMA) induced mRNA expression of the glutathione S-transferase alpha 1 gene (Gsta1) located downstream of the cis-acting anti-oxidant responsive element (ARE). Herein, we tested the hypothesis that MMA activated the Gsta1 promoter through the ARE. HepG2 cells were transfected with a luciferase reporter vector containing the ARE and the Gsta1 promoter (−990 to +46 bp) and cultured for 12 hrs with MMA (initial concentration, 10 mM). Analysis of the expressed luciferase activity indicated that MMA activated the promoter 2.6-fold. MMA (from 1 to 30 mM) dose-dependently increased the promoter activity, which reached a plateau between 6 and 12 hrs. In HepG2 cells transfected with a reporter vector containing 2 AREs and a TATA-like promoter, 10 mM MMA increased the reporter expression 2.8-fold. These results suggest that MMA increases Gsta1 transcription through ARE-mediated promoter activation.

The effect of zinc levels in a gold-based alloy on porcelain–metal bonding

OBJECTIVES The purpose of this study was to ascertain whether the amount of Zn in gold alloys contributes to porcelain-metal bonding. METHODS Experiments were carried out using a commercial Pd-free gold alloy with a nominal composition of 88.7 wt% Au, 9.49 wt% Pt, 1.5 wt% Zn, 0.1 wt% Mn, 0.2 wt% Rh, and 0.1 wt% Ir, which contains Zn and no other elements (In, Sn, Fe) known to affect porcelain-metal bond strength. To establish the effect of oxidation of the metal surface, porcelain was applied both to preoxidized and to non-preoxidized metal specimens. The bond strength was evaluated by means of the ISO 9693: 1999 crack initiation test. A conventional gold alloy was used as a control. The elemental distributions at the porcelain/alloy interfaces were analyzed in cross section by electron probe microanalysis. Additionally, after the bond strength test, cross-sections of the interfaces of the debonded specimens were microscopically analyzed to characterize the fracture mode. RESULTS The Pd-free gold alloy joints showed significantly higher bond strength values than joints made with conventional gold alloy. Preoxidation treatment significantly increases the bond strength, in the preoxidized joints Zn was highly localized at the interface and diffused into the porcelain up to about 10 μm from the interface, and the joint failed by cohesive fracture in the porcelain. In contrast, the non-preoxidized joint showed mainly adhesive fracture at the porcelain/alloy interface. SIGNIFICANCE The presence of Zn in gold alloys plays a part in establishing chemical bonding thus improving the bond strength between porcelain and alloy.

The Biological Properties of a Novel Ethyl Methacrylate Resin

A novel ethyl methacrylate (EMA) resin was developed to overcome the tissue, organ and systemic damage associated with the residual monomer of conventional methyl methacrylate (MMA) resin bone cement. EMA resin is a chemical/ photopolymerizable material and is easy to handle during clinical procedures. The biocompatibility of EMA was evaluated in accordance with ISO10993-6. No inflammatory response was observed 1 and 9 weeks after implantation in the dorsal subcutaneous tissue of ddY mice. EMA resin also demonstrated better biocompatibility when compared with conventional bone cements. Poly-l-lactic acid (PLLA) was used as a carrier for bone morphogenetic protein (BMP) and added to the EMA slurry. The EMA-PLLA composite membrane was sticky and BMP readily adhered to its surface. The EMA-PLLA-BMP composite membrane induced new bone formation, the new bone growing in the shape of the EMA in the thigh muscle pouch of ddY mice. This novel EMA resin has many potential clinical applications.

Bone morphogenetic protein-induced heterotopic bone in osteopetrosis.

The objectives of the present research on the osteopetrotic mouse are to investigate the factors influencing heterotopic bone development. The osteopetrotic mutant was deficient in macrophage colony stimulating factor and failed to activate functioning monocytes, macrophages, and osteoclasts. Macrophage colony stimulating factor deficiency also caused a heretofore undescribed delay in organization and absorption of hematomas resulting from surgical operations. Surgically implanted in a heterotopic site, bone morphogenetic protein induced approximately 10% more bone in osteopetrotic than littermate +/? mice. Radiographically, the heterotopic bone was at least 50% denser than new bone. The new bone was metachromatic or slightly basophilic rather than eosinophilic and undermined with large deposits of hypercalcified hypertrophic cartilage. Bone mineral in the osteopetrotic mouse was deposited in an apatite-like form with a higher calcium/phosphorus ratio than the bone of +/? littermates. High levels of alkaline phosphatase synthesis were sustained longer in the osteopetrotic mouse than in the +/? littermate. Tartrate resistant acid phosphatase synthesis was almost nil in osteopetrotic mice during the first 4 weeks, and thereafter appeared coincidental with spontaneous remission of osteopetrosis at 6 weeks. Implants of the mineralized cortical bone matrix of the osteopetrotic mouse showed minimal if any bone morphogenetic protein activity of matrix of +/? littermate or otherwise normal mice. The cause of the remission of the bone disorder in the osteopetrotic mouse is not known but is of great interest to students studying the problem of coupling of bone formation to bone resorption.

A lysyl oxidase distinct from amine oxidase in bovine dental pulp

Abstract The activity of lysyl oxidase, which catalyzes the oxidation of peptidyl lysine in collagen or elastin, was negligible in a salt extract from bovine dental pulp. However, an extract with salt and 4 M urea contained significant enzyme activity. In contrast, considerable amine oxidase activity, with benzylamine or kynuramine as substrate, was present in the salt extract either with or without 4 M urea. The activities of lysyl oxidase and amine oxidase were completely separated by Sephadex G-200 column chromatography. It is concluded that the lysyl oxidase and amine oxidase are distinct enzymes.