Near-threshold fatigue crack propagation without oxide-induced crack closure.

An accurate value for the threshold stress intensity factor range, ΔKth, is a key parameter for studying crack-like defects. However, it is difficult to obtain accurate ΔKth values due to oxide-induced crack closure. In this study, we report conditions for minimizing the effects of oxide-induced crack closure near the threshold region, where a concave curve of the effect on the loading frequency on oxide-induced crack closure is achieved. The resulting conditions allow for an accurate determination of ΔKth, which is a key material parameter relating to the pertinent loading ratio.

A two year in vivo study of polyvinyl alcohol-hydrogel (PVA-H) artificial meniscus.

For the recognized importance of knee meniscus function, the treatment of meniscus injury has been changing from resection to repair. However, depending on the type of injury, meniscectomy sometimes cannot be avoided. In such a case, it is important to anticipate the future problem of degenerative change or osteoarthrosis in the knee joint. In consideration of the prognosis and circumstances in such patients, we have developed an artificial meniscus using polyvinyl alcohol-hydrogel (PVA-H) for salvage. We have already reported the results up to 1 year after animal operation. The present study investigated the results in postoperative 2.0 years to assess further the use of artificial meniscus. In the results, the articular cartilage state of knee joint implanted PVA-H meniscus was good even after 2 years, while Osteoarthrosis (OA) change progressed in meniscectomy knee joint. In addition, neither wear, dislocation nor breakage of PVA-H was observed. These results proved that an artificial meniscus using PVA-H can compensate for meniscal function and might be clinically applicable.

P450 aromatase inhibition assay using a competitive ELISA.

P450 aromatase (P450arom) is a well known target by anti-cancer drugs and toxic chemicals and efficient and convenient analytical tools are desired for. We established a convenient assay for P450arom inhibition based on an enzyme-linked immunosorbent assay (ELISA). The first step of the assay consists of a P450arom reaction, which converts a testosterone to a 17beta-estradiol using a recombinant human P450arom and a NADPH regenerating system. The second step of the assay consists of an ELISA system using a highly specific and sensitive anti-estradiol monoclonal antibody in conjunction with estradiol-3-CMO-horseradish peroxidase (E2-3-CMO-HRP). This system has advantages over other P450arom assays because it does not use radioactive ligands and because it is not subject to interference from self-fluorescing test compounds. We could successfully estimate some types of P450arom inhibitors reported before. This assay should be very useful for high throughput screening of drug candidates and endocrine disrupting chemicals via P450arom.

Preliminary study of polyvinyl alcohol-hydrogel (PVA-H) artificial meniscus.

Nowadays the importance of knee meniscal function is recognized. The treatment for meniscus injury has been changing from resection to repair. However, depending on the type of injury, meniscectomy cannot be avoided. In consideration of the prognosis in such patients, we developed artificial meniscus using polyvinyl alcohol-hydrogel (PVA-H) with a high water content and performed an animal experiment as preliminary study. In the experiment using rabbits, the lateral meniscus was replaced with an artificial meniscus in one knee side and lateral meniscectomy was performed in another knee side of each rabbit. In the knees treated by artificial meniscus replacement, regressive changes were initially observed but did not progress after a certain period, and the articular cartilage state was good even after 1 year. In addition, neither wear nor breakage of PVA-H was observed. These results suggest that artificial meniscus using PVA-H with a high water content compensates for meniscus function and is clinically applicable. However, for clinical application some problems such as fixation method, tolerance of PVA-H, remain to be solved.

Use of controlled mechanical stimulation in vivo to induce cartilage layer formation on the surface of osteotomized bone.

A micromachine was used to study the response of mesenchymal tissue to mechanically controlled motion in vivo. The middle portion of the coccygeal vertebra of Fischer 344 rats was osteotomized, and continuous bending motion was applied for 4 weeks. The experimental groups were divided into two groups with higher sliding displacement applied at the osteotomized gap of group II. Hyaline cartilage tissue was generated at the osteotomized ends, and was predominantly formed on the side that extended during the bending motion. These newly formed tissues stained intensively with safranin O and toluidine blue, positively with immunostain for type II collagen, but negatively with immunostain for type I collagen. Articular cartilage-like tissues with a surface and a layer structure were obtained in group II, in which higher sliding motion was applied. Light and electron microscopy revealed morphological features similar to those of normal articular cartilage tissue in the superficial and middle zones of the tissues obtained in group II. Collagen fibrils in the superficial zone were found aligned parallel to the smooth surface. Although tidemark formation was not observed in the deep zone, the structure was much more natural than that of any other tissue-engineered cartilage reported to date. These results suggest that controlled sliding stimulation can elicit the generation of articular cartilage structure in vivo.

30 mm regeneration of rat sciatic nerve along collagen filaments.

This paper describes 30 mm regeneration of peripheral nerve axons along collagen filaments; 31-mm-long collagen filaments or collagen tube were grafted to bridge a 30-mm defect of rat sciatic nerve. The mean number and the diameter of regenerated myelinated axons were 330+/-227 and 2.7+/-0.9 microm at the distal end of the collagen-filaments 12 weeks postoperatively; while at the distal end of the tube no axon was found.

Bridging a 30-mm nerve defect using collagen filaments.

This article describes a 30-mm regeneration of severed peripheral nerve axons along collagen filaments. Two thousand or 4000 31-mm-long collagen filaments were grafted to bridge a 30-mm defect of the rat sciatic nerve. A collagen tube was grafted as a control. The mean number and mean fiber diameter of regenerated myelinated axons were 330 +/- 227 and 2.7 +/- 0.9 microm in the distal end of the 2000 collagen-filaments nerve guide, and 564 +/- 275 and 2.5 +/- 1.1 microm in the distal end of the 4000 collagen-filaments nerve guide at 12 weeks postoperatively, whereas in the distal end of the collagen tube, no regenerated axon was found. These results suggest that the collagen filaments guide axons of the rat's sciatic nerve to regenerate for 30 mm and act as a scaffold for axonal regeneration. Thirty-millimeter nerve regeneration of a 1-mm-diameter rat sciatic nerve by an artificial nerve guarantees a clinical application of the implant which should be very important for patients and surgeons.

Restoration of function after spinal cord transection using a collagen bridge.

The restoration of function of transected adult mammalian spinal cord without living tissue has not been reported previously. We report the first success of functional restoration of transected spinal cord without living tissue. We grafted collagen filaments parallel or transverse to the axis of the spinal cord to bridge 5-mm defects of 47 adult rat spinal cords. Twenty-five rats were used as a control. Of the 72 rats, 42 rats survived the experimental period. At 4 weeks postoperatively, regenerated axons crossed the proximal and distal spinal cord-implant interfaces in all 5 rats of the parallel-grafted group. At 12 weeks postoperatively, the rats in the parallel-grafted group (8 rats) could walk, run, and climb with hind-forelimb coordination. The somatosensory-evoked potentials were seen. Results suggest that the collagen filaments support the axonal regeneration of the transected spinal cord and the restoration of function when grafted parallel to the axis of the spinal cord. The functional restoration appeared to be permanent, raising the possibility of therapeutic application in humans.

Nerve regeneration along collagen filament and the presence of distal nerve stump.

This article describes the regeneration of severed peripheral nerve axons along collagen filaments in the absence of the distal nerve stump. 22-mm long nerve guides made of collagen filaments were sutured to the proximal ends of severed rat sciatic nerves. The distal ends of the guides were sutured to the distal stumps of the nerves in a group and not sutured in the other. Nerve autografts and collagen tubes were used as controls. At 8 weeks postoperatively, the mean number and the mean diameter of myelinated axons were 5491 +/- 617 (mean +/- SD) and 2.3 +/- 1.3 microns at the distal ends of the collagen filaments nerve guides those the distal ends were sutured to the distal stumps of the nerves, while in the nerve autografts these were 4837 +/- 604 and 3.3 +/- 1.4 microns. These were 1992 +/- 770 and 2.7 +/- 1.2 microns at the distal ends of the collagen-filaments guides those the distal ends were not sutured to the distal stumps of the nerves, while in the nerve autografts these were 3041 +/- 847 and 2.3 +/- 1.1 microns. No axon was found at the distal ends of the collagen tubes. The results suggested that the contact guidance and the chemotaxis guided regenerating axons along the collagen filaments.

Attachment of artificial cartilage to underlying bone.

The key problem with artificial joint materials is obtaining quick and firm attachment onto the underlying bone. In developing artificial articular cartilage, composed of polyvinyl alcohol hydrogel (PVA-H), this problem was solved by using a composite osteo-chondral device (COD). This enables attachment within four weeks post-operation by massive bone ingrowth into the pores. The COD consists of PVA-H as an artificial cartilage and titanium fiber mesh (TFM) as porous artificial bone. In this study, the strength of the shear resistance force at the interface of the PVA-H and the TFM fabricated by injection molding and the changes in the mechanical properties of the PVA-H fabricated by high temperature during the injection-molding process were examined. The shear resistance force was strengthened markedly by using injection molding and no important deterioration of the PVA-H was found. Morphological examination of canine spines, to which artificial intervertebral discs made of the COD were implanted, showed good bonding of the COD with the vertebral bodies for an extended period of 30 months, and encouraged us to use the COD clinically.

Host-dependent activation of IS1203v excision in Shiga toxin-producing Escherichia coli.

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.

Significance of peak value and duration of the interfacial shear load in evaluation of the bone-implant interface.

OBJECTIVE: To determine if there is any merit in measuring peak load duration in studies of bone-implant interface during a pull-out test was performed to record the interfacial shear load. DESIGN: Mechanical and histological investigations were performed to determine the bone-implant interfacial strength. BACKGROUND: A common long-term problem following artificial joint replacement is the formation of an intervening fibrous tissue seam at the interface between bone and a prosthesis. To investigate the nature and strength of interfacial attachment between bone and an implanted biomaterial, a detaching method such as pull-out test can be used. METHODS: Four porous surfaced alumina ceramic implants with or without glass-ceramic containing apatite and wollastonite coating at the surface of the beads and the substrate were implanted into canine the femoral condyles. Peak value and its duration of the interfacial shear load for the different types of implants were analyzed. Tissue formed at the bone-implant interfaces were examined histologically.Results. The peak value of interfacial shear load of the apatite and wollastonite glass-ceramic-coated implant was significantly greater than that of the non-coated implant at every stage, but the peak value duration in the control at each time period was greater than that of the coated implant. Histologically, more bone tissue ingrowth in the pores of coated implants, whereas a gap was observed at the interface of bone and the non-coated implant. CONCLUSIONS: The interfacial shear load values, peak and its duration, are significantly influenced by the direct bone bonding or the intervening fibrous tissue which formed at the bone-implant interface. RELEVANCE: The peak load measurement and the duration of the peak load value are useful to measure the strength of interfacial attachment between bone and an implanted biomaterial. However, the duration of peak load value is closely related to the strength and plasticity of the tissue formed in the bone-implant interface. The analysis of peak value duration could be used to determine the properties of new grown tissue before histologic examination.

Development of an artificial meniscus using polyvinyl alcohol-hydrogel for early return to, and continuance of, athletic life in sportspersons with severe meniscus injury. I: mechanical evaluation.

The importance of knee meniscus function is now recognized, and the treatment of meniscus injury has been changing from resection to repair. However, depending on the type of injury, meniscectomy sometimes cannot be avoided. The young athlete might undergo meniscectomy in order to return to sports life as early as possible. However, in such a knee, it is important to anticipate the future problem of degenerative change or osteoarthrosis. In consideration of the prognosis and circumstances in such patients, we have developed an artificial meniscus using polyvinyl alcohol-hydrogel (PVA-H) and performed mechanical tests for compression and stress-relaxation. We found that the human meniscus has unique viscoelastic properties and a high water content. PVA-H showed viscoelastic behaviour similar to that of human meniscus in mechanical tests. These results suggest that an artificial meniscus using PVA-H with a high water content can compensate for meniscal function and might be clinically applicable.

Development of an artificial meniscus using polyvinyl alcohol-hydrogel for early return to, and continuance of, athletic life in sportspersons with severe meniscus injury. II: animal experiments.

Due to the prognosis and sporting needs of young athletes who have severe meniscus injury, we have developed an artificial meniscus using polyvinyl alcohol-hydrogel (PVA-H). Biomechanical studies have shown that the human meniscus has various mechanical functions; in our mechanical tests, specimens of human meniscus (part I of this study) demonstrated unique viscoelastic properties. Those tests also showed that a high water-content PVA-H has very similar mechanical properties to those of human meniscus and potential as a substitutive meniscus implant. To assess further the use of PVA-H as an artificial meniscus, we have now performed animal experiments using a PVA-H with 90% water content. In rabbits, the lateral meniscus was replaced with an artificial meniscus in one knee of each animal and lateral meniscectomy was performed on the other side as a control. In the knees of meniscectomy group, degenerative changes in articular cartilage progressed with time and osteoarthritis was observed after 1 year. In the knees of the artificial meniscus group, regressive changes were initially observed, but did not progress, and knee joint function was good even after 1 year. Neither wear nor breakage were observed in artificial menisci. These results suggest that an artificial meniscus using a high water-content PVA-H might be clinically applicable.

The lubricative function of artificial joint material surfaces by confocal laser scanning microscopy. Comparison with natural synovial joint surface.

The purpose of this study was to observe and compare the effect of the behavior of different lubricating surfaces, including articular cartilage and several artificial joint materials, under the physiological loading by confocal laser scanning microscopy (CLSM) to clarify the mechanism of lubrication in natural joints and subsequently improve the quality of artificial joints. In our experiment, even with considerable loading, natural articular cartilage exhibited a synovial fluid area and an area of direct and solid contact. In the region between these two areas, a liquid crystal layer was observed. On the other hand, the materials used for artificial joints (metal and polyethylene, which are now in use, and polyvinyl alcohol-hydrogel polymer which is being developed), did not exhibit neither a clear fluid pool area nor the intermediary area with liquid crystal formation. These results suggest that natural articular cartilage surface has a particular characteristic which builds up a synovial pooling area and liquid crystal formation in the third area by interaction with macromolecules in synovial fluid under the loading condition. These characteristics give natural articular cartilage its excellent lubricative function. To improve the quality of artificial joints, the characteristics of the implant material surface and the synovial macromolecules must be considered.

Functional similarities of a thermostable protein-disulfide oxidoreductase identified in the archaeon Pyrococcus horikoshii to bacterial DsbA enzymes.

We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH(2)-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH(2)-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.

Characterization of a polyvinyl alcohol-hydrogel artificial articular cartilage prepared by injection molding.

We have developed a hip hemi-arthroplasty using polyvinyl alcohol-hydrogel (PVA-H) as the treatment for hip joint disorders in which the lesion is limited to the joint surface. In previous studies, we characterized the biocompatibility and the mechanical properties of PVA-H as an arthroplasty material. To fix PVA-H firmly to the bone, we have devised an implant composed of PVA-H and porous titanium fiber mesh (TFM). However, because of poor infiltration of the PVA solution into the pores of the TFM when using the low temperature crystallization method, the strength of the PVA-H-TFM interface was insufficient. Consequently, the infiltration method was improved by adopting high-pressure injection molding. With this improved method, the bonding strength of the interface increased remarkably. However, as this injection molding requires high temperature, various mechanical properties of the PVA-H might change with this treatment in comparison with the previous method. The purpose of this study was to investigate the effect of high temperature treatment on the mechanical properties of PVA-H as artificial articular cartilage, the tensile test and friction test were performed about new PVA-H. The results showed no significant mechanical deterioration of the PVA-H. This certified that the injection-molding method did not induce the change of the mechanical properties of PVA-H and indicated the potential of hemi-arthroplasty using PVA-H by this method in the future.

Composite device for attachment of polyvinyl alcohol-hydrogel to underlying bone.

Polyvinyl alcohol-hydrogels (PVA-H) have been specifically proposed as promising prosthetic biomaterials because of their excellent mechanical properties and biocompatibility. However, it is very difficult to obtain stable long-term fixation of implanted PVA-H and this problem is the key point to be solved in the further development of PVA-H in biomedical applications more widely in various fields. Orthopedic implants using PVA-H have been developed and in the process this problem was solved using a composite device. This device consists of PVA-H and a titanium fiber mesh (TFM) as a porous tissue-ingrowth material. In the present study, the mechanical properties of this composite device produced using a new improved production method (injection molding) were investigated and animal experiments for in vivo evaluation were performed. In vitro tests showed that the shear strength at the interface of the PVA-H and TFM, and the mechanical properties of PVA-H itself were promising. Moreover, the histological appearance of the composite device implant in vivo showed abundant bone ingrowth into the deep pore spaces of TFM, which indicated that this device attached firmly to the adjacent bone. These results indicated this composite device prepared by this new method can supply firm attachment of PVA-H onto underlying bone, and that it will be available for various biomedical applications using PVA-H.

Double evaluations of chemicals using a cocktail of fused recombinant receptors.

Some chemicals have multipotential as endocrine-disrupting chemicals (EDCs). For example, some chemicals act as both estrogens and antiandrogens. Numbers of such chemicals should be evaluated from many aspects; however, labor and expenses are generally limited. We have developed two expression systems for the wild type of human estrogen receptor alpha and the wild type of human androgen receptor fused with a maltose binding protein. They are soluble and have binding activities. They showed dose-responses to natural hormones and well-known potential EDCs. After we established each assay condition for a competitive binding assay using each receptor, we found that two assay systems can be carried out simultaneously under limited and harmonized conditions. Under harmonized conditions using a cocktail of two types of receptors, we could estimate natural hormones and potential EDCs. Interference between two assay systems was not observed under these conditions. We believe that some competitive binding assays can be carried out using a cocktail of receptors at the same time if interference among different assay systems can be avoided by choosing ideal conditions.

Bridging a spinal cord defect using collagen filament.

STUDY DESIGN: A rat model of spinal cord defect was designed to evaluate the effect of collagen filament implant on nerve regeneration in the spinal cord defect. OBJECTIVES: To bridge a spinal cord defect and restore the function in adult mammals. SUMMARY OF BACKGROUND DATA: Resection of the spinal cord in mammals is always followed by motor paralysis and loss of voluntary function below the lesion. Partial success in bridging the ends of the spinal cord after complete resection was reported. However, restoration of function has not been reported in adult mammalian. MATERIALS AND METHODS: Four thousand collagen filaments 5-mm-long were grafted to bridge a 5-mm defect of rat spinal cord. Controls had their spinal cord defect left ungrafted after resection. At 1-week intervals, animals were evaluated functionally. After 4 and 12 weeks, animals were evaluated histologically. After 12 weeks, animals were evaluated electrophysiologically. RESULTS: The severed spinal cord axons regenerated along the collagen filament implant crossing the proximal and distal spinal cord implant interfaces at 4 weeks after surgery. The rats with collagen filament grafts could walk, run, and climb with hind forelimb coordination at 12 weeks after surgery. Sensory-evoked potential waveform was found in the rats with collagen filament at 12 weeks after surgery. CONCLUSIONS: The collagen filaments support the axonal regeneration of the transected spinal cord and the restoration of function.