The chemical basis for the ferriprotoporphyrin IX-chloroquine complex induced lipid peroxidation.

Ferriprotoporphyrin IX (FPIX) forms a coordination complex with chloroquine, an anti-malarial drug. The FPIX-chloroquine complex strongly promotes the peroxidative cleavage of phospholipid membrane. Iron in the complex is essential for the complex to induce lipid peroxidation. In this paper a more detailed mechanism of the complex promoted lipid peroxidation was investigated. Apotransferrin exhibited no apparent inhibition of the complex evoked lipid peroxidation, indicating no mobilization of iron from the complex. No significant inhibitory effect by superoxide dismutase, catalase and sodium benzoate on the complex induced lipid peroxidative reaction, suggesting little involvement of superoxide anion, hydrogen peroxide and hydroxyl radical in the reaction. Quinine and mefroquine, blood shizontocidal drugs as well as chloroquine, formed a complex with FPIX and each complex more rapidly induced lipid peroxidation than FPIX alone. Primaquine, which is not as effective as quinine or mefroquine on an intraerythrocytic malaria parasite, neither coordinated to FPIX nor promoted lipid peroxidation. The complex formation between FPIX and chloroquine, quinine or mefroquine could play a key role in their anti-malarial actions.

Re-evaluation of the biocompatibility of bioinert ceramics in vivo.

The affinity of bone for bioinert ceramics and stainless steel was compared using calcified bone specimens. We implanted cylinders of alumina ceramics (Al2O3), zirconia ceramics (ZrO2) and SUS-316 L stainless steel into the distal femoral epiphyses of dogs and then made observations from 4 to 96 wk post-operatively. Irregularities in the histological specimens suggested the presence of artefacts due to the insertion technique. We subsequently used screws inserted into holes tapped with a tap that had a diameter identical to the screws, and observed these implants from 4 to 96 wk after insertion. There was no detectable difference in the affinity index for all three materials from 4 to 96 wk after implantation. The affinity index was calculated as the ratio of the new bone directly adjoining the implant without any intervening fibrous membrane or bone marrow to the total length of the bone-implant interface x 100%.

Remodelling of bone around hydroxyapatite and titanium in experimental osteoporosis.

The affinity of the tibia bone for cylinders of hydroxyapatite (HA) and Ti-6Al-4V was investigated in three experimental animal models: intact rat tibiae, ovariectomized rat tibiae and ovariectomized plus neurectomized rat tibiae. The affinity index (the length of bone directly apposed to the implant/the total length of the bone-implant interface x 100%) was calculated. In intact and ovariectomized tibiae, no significant difference existed between the affinity index of HA and Ti-6Al-4V. In ovariectomized plus neurectomized tibiae, the affinity index of HA was greater than for Ti-6Al-4V from 8 to 24 wk after implantation (8 wk, P < 0.05; 12 wk, P < 0.005; 24 wk, P < 0.05). The affinity index of Ti-6Al-4V in normal tibiae was greater than that in ovariectomized or ovariectomized plus neurectomized tibiae. There was no significant difference of the affinity index of HA between normal, ovariectomized or ovariectomized plus neurectomized tibiae. Osteoporotic bone demonstrated a greater affinity to HA than Ti-6Al-4V in an experimental animal model.

Bone-implant interface mechanics of in vivo bio-inert ceramics.

We have previously demonstrated that there was no significant difference between the affinity of bone to bio-inert ceramics and stainless steel in a histological study. In this study, the bone-implant interface shear strength of alumina ceramics (Al2O3), zirconia ceramics (ZrO2), stainless steel (SUS316L) and sintered hydroxyapatite (HA) were compared in 19 dogs using a transcortical push-out model of the femur 4 and 12 wk after implantation. The interface shear strength of HA was significantly greater than that of alumina ceramics, zirconia ceramics and stainless steel (P < 0.001). There was no significant difference between bio-inert ceramics and stainless steel.

Surgical biomaterials and differential colonization by Staphylococcus epidermidis.

The data presented in this communication demonstrate preferential colonization of certain biomaterials by Staphylococcus epidermidis. Using a laminar flow biomaterial colonization chamber and surgical-grade biomaterials (stainless steel, aluminium ceramic, methyl methacrylate and high-density polyethylene), the pattern of colonization was quantitated using plate count techniques and electron microscopy. Under comparable conditions, methyl methacrylate was colonized by S. epidermidis in greater numbers than the other biomaterials. Increased bacterial colonization and slime production on methyl methacrylate was time-dependent and 15 times higher than on stainless steel and aluminium and four times higher than on high-density polyethylene. The data reveal that certain biomaterials may promote infection by favouring colonization by potential pathogens. This variable should be explored extensively in an in vivo setting because of its implication in clinical infections.

Evaluation of the risk of instrumentation as a foreign body in spinal tuberculosis. Clinical and biologic study.

The risk of persistence and recurrence of infection in posterior spinal instrumentation surgery for spinal tuberculosis was studied clinically and microbiologically. Eleven patients with thoracic, thoracolumbar, and lumbar spinal tuberculosis treated by debridement, anterior fusion, and combined posterior instrumentation surgery were analyzed. Seven patients had tuberculosis in both anterior and posterior spinal elements. There were no cases of persistence or recurrence of infection after surgery, and instrumentation provided immediate stability and protected against development of kyphotic deformity. The adherence properties of Mycobacterium tuberculosis to stainless steel (SUS 316) was evaluated experimentally. The results showed that posterior instrumentation surgery was not a hazard to spinal tuberculosis infection when combined with radical debridement and intensive anti-tuberculosis chemotherapy.

Use of a reconstituted basement membrane to study the invasiveness of tumor cells.

We have used an extract of basement membranes which can be reconstituted into a biologically active gel matrix composed predominantly of collagen IV, laminin, nidogen, and heparin sulfate proteoglycan, in order to study the mechanisms involved in tumor cell invasion. When layered onto a porous filter in a Boyden chamber, the gel forms a barrier to the passage of normal cells. Malignant cells are able to cross this layer when the conditioned medium of NIH 3T3 cells is used as a chemoattractant to stimulate cell migration. A variety of human tumor cells have thus been studied in this system and we find a high correlation between their invasiveness in vitro and their malignant behavior as exhibited in vivo. We have used this in vitro invasion assay to test for factors which might inhibit tumor cell invasion. Collagenase IV is produced by malignant cells and is thought to be required for invasion. Indeed, inhibitors of this enzyme have demonstrated reduced tumor cell invasiveness. One site of five amino acids, on the B1 chain, which has been shown to promote cell adhesion, migration and binding to laminin receptor, was found to inhibit the invasion of tumor cells. In addition, factors which elevated cAMP levels were also able to suppress the invasiveness of tumor cells. These data suggest that the assay system described herein can be successfully utilized to study the invasive activity of tumor cells and those factors that may inhibit the spread of malignant cells.

The inhibition of bacterial adhesion to a tobramycin-impregnated polymethylmethacrylate substratum.

Tobramycin sulfate powder (1.2 g) was mixed with Palacos polymethylmethacrylate (PMMA) bone cement (40 g) to produce 100 discs containing 5.9 mg tobramycin per disc. These discs were used to evaluate the inhibition of bacterial adhesion to an antibiotic-laden biomaterial. Tobramycin-impregnated PMMA discs and control discs containing no tobramycin were exposed in vitro to Staphylococcus epidermidis. Colonization was quantitated using plate count techniques and electron microscopy. Tobramycin-impregnated surfaces reduced adhesive bacteria colonization by 1 log relative to control discs. These observations suggest that tobramycin-impregnated PMMA may not be significantly effective in preventing colonization of the biomaterial substratum and PMMA may be a poor choice as a drug delivery vehicle in biomaterial and compromised tissue-centered infections.

Increased resistance of bacteria after adherence to polymethyl methacrylate. An in vitro study.

The pathobiology of total joint prosthesis infection was investigated in vitro. Discs of polymethylmethacrylate (PMMA) were exposed to a suspension containing cells of 10(8) per mL Staphylococcus epidermidis E-46. After 12 hours, exposed discs were rinsed with phosphate-buffered saline and placed in brain heart infusion broth containing antibiotics (2.5 mg per mL of Cephaloridine). After gentle shaking for 24 hours at 37 degrees C, the bacteria on the PMMA surface were detached and washed with phosphate-buffered saline to remove the antibiotics. Compared with the free bacteria which were detached from the PMMA by sonication immediately after exposure to the antibiotic solution, those allowed to remain adhered to the PMMA surface were more resistant to antibiotics. Scanning electron microscopy showed accumulation of bacteria surrounded by slime on PMMA discs exposed for 12 hours. Our results indicate that resistance of bacteria to antibiotics is increased after adherence to the biomaterial and formation of a slime layer.

Inhibition of bacterial adhesion by tobramycin-impregnated PMMA bone cement.

We investigated the effect of tobramycin-impregnated polymethylmethacrylate (PMMA) bone cement on the adhesion and colonization of Staphylococcus epidermidis. The pattern of colonization was quantitated using plate count techniques and electron microscopy. Colonization of the tobramycin-impregnated disc surface by adhesive bacteria was demonstrated but it was less than in the control disc. This finding suggests that tobramycin may reduce bacterial adherence and proliferation on the PMMA surface.

Bacterial adherence to bioinert and bioactive materials studied in vitro.

In vitro, bioinert stainless steel and titanium alloy, and bioactive sintered hydroxyapatite and hydroxyapatite-coated titanium materials were exposed to Staphylococcus epidermidis to study bacterial adhesion. Scanning electron microscopy showed that fibrous strands interconnected the adherent bacteria, and that background matrix enclosed bacterial colonies. This adherent mode of growth may reduce the susceptibility of the bacteria to host clearance mechanisms and antibiotic therapy. Adherence assays revealed that bacterial adherence to sintered hydroxyapatite was higher than to the other 3 materials.

Quantitative analysis of in vivo tissue responses to titanium-oxide- and hydroxyapatite-coated titanium alloy.

We have previously studied a large number of histological specimens of biomaterials and found that regions with and without an intervening fibrous membrane coexisted in many specimens. Therefore, it appears necessary to perform an evaluation of the entire specimen when histologically assessing the affinity of bone for a biomaterial. Accordingly, we performed a quantitative histological evaluation of hydroxyapatite (HAP)- and titanium-oxide(TiO2)-coated Ti-6A1-4V and uncoated Ti-6A1-4V (control) by determining the affinity index. This was defined as the length of bone directly opposed to the implant/the total length of the bone-implant interface X 100%. The test materials were inserted into the distal epiphyseal region of the femurs of adult dogs, and follow-up quantitative histological comparisons were performed from 4 weeks to 96 weeks. The HAP-coated implants had the highest affinity index 4 weeks after insertion, and this superiority was maintained up to 96 weeks. There was a significant difference in affinity index between HAP-coated implants and control implants (P less than 0.001), while TiO2-coated implants showed no significant difference in comparison to the control.

Comparison of bone-implant interface shear strength of solid hydroxyapatite and hydroxyapatite-coated titanium implants.

The interface shear strength of uncoated Ti-6Al-4V, dense sintered hydroxyapatite (HA), and HA-coated Ti-6Al-4V were compared. Interface shear strength was determined using a transcortical push-out model in dogs 4 and 12 weeks after implantation. The interface shear strength of dense sintered HA and HA-coated Ti-6Al-4V was significantly higher than that of uncoated Ti-6Al-4V (P < .001). There was no significant difference between the interface shear strength of dense sintered HA and HA-coated Ti-6Al-4V. After the push-out test for HA-coated implants, the regions fractured at the bone-coating interface and at the coating-titanium interface coexisted at 4 weeks after implantation. At 12 weeks, the fracture site was, in all cases, the HA coating-titanium interface, and, in a few samples, fractures inside the coating layer also were visible.

Comparison of bone-implant interface shear strength of hydroxyapatite-coated and alumina-coated metal implants.

We performed a transcortical push-out test to determine the effect of surface roughness of hydroxyapatite (HA)-coated implants on bone-implant shear strength in a canine model. Hydroxyapatite- and alumina-coated SUS316L with the same surface roughness (roughness average: Ra = 5 microns) and HA-coated Ti-6A1-4V (Ra = 8.4 microns), sintered HA (Ra = 0.9 micron), and dense alumina (Ra = 1.3 microns) were inserted into the dog's femur. The interface shear strength of the dense alumina was significantly lower than that of other implants at both 4 and 12 weeks after implantation. At 4 weeks after implantation, the interface shear strength of the alumina-coated SUS316L was significantly lower than that of other implants (P < .05) except the dense alumina, but at 12 weeks, there was no significant difference between the implant types except the dense alumina. This indicates that the surface roughness of the HA coating affects the enhancement of the bone-implant interface shear strength at the early period after implantation, and that a surface roughness of several micrometers does not influence the bond strength between bone and HA. A scanning electron microscopic study indicated that in almost all cases at 12 weeks, the failure site after push-out testing was the coating-substrate interface, not the coating-bone interface. Therefore, protection of the coating-substrate interface from direct shear loading is needed.

Evaluation of carcinogenicity and chronic toxicity associated with orthopedic implants in mice.

The carcinogenicity and chronic toxicity of 316L stainless steel, nickel, Ti-6A1-4V, hydroxyapatite (HA)-coated Ti-6A1-4V, aluminum oxide containing yttrium oxide, and zirconium oxide containing yttrium oxide were evaluated by implanting solid rods of each material in the thigh muscle of C57BL/6N mice for 24 months. Nickel alloy showed high carcinogenic and toxic potencies, whereas other materials showed no evidence of them. Tumors retaining nickel alloys were malignant fibrous histiocytoma or fibrosarcoma. In some cases, lymphomata that seemed to develop spontaneously were found around the implants because lymphocytes were known to accumulate in chronic inflammatory lesions, and this phenomenon also might be applied to lymphoma.

Case report 877: Giant cell reparative granuloma arising in enchondromatosis.

A case of giant cell reparative granuloma in a 16-year-old girl has been reported. This is the first reported case of giant cell reparative granuloma associated with enchondromatosis. In addition to the clinical features, the pathologic findings and differential diagnosis were also discussed.

Evaluation of metal implants coated with several types of ceramics as biomaterials.

The in vivo biocompatibility of metals coated with several different types of ceramics [alumina (alpha-Al2O3), titanium oxide (TiO2), titanium nitride (TiN), and hydroxyapatite (HAP)] was investigated. These composites had been devised for the purpose of incorporation into the stem of a total hip prosthesis. The materials were inserted into the mid-diaphyseal region of the femurs of adult dogs, and follow-up quantitative histological comparisons were performed for a period of up to 96 weeks. HAP-coated composites showed the best biocompatibility.

Hydroxyapatite-coating on titanium arc sprayed titanium implants.

We developed a new titanium spray technique using an inert gas shielded arc spray (titanium arc spray). Hydroxyapatite (HA)-coating can be applied to the implant without any surface pore obstruction after the rough surface is made by this technique. Scanning electron microscopy (SEM) of various porous implant surfaces after HA-coating revealed that the bead and fiber metal-coated implants had either a pore obstruction or an uneven HA-coating. On the other hand, the titanium arc sprayed implant demonstrated an even HA-coating all the way to the bottom of the surface pore. In the first set of animal experiments (Exp. 1), the interfacial shear strength to bone of four kinds of cylindrical Ti-6A1-4V (Ti) implants were compared using a canine transcortical push-out model 4 and 12 weeks after implantation. The implant surfaces were roughened by titanium arc spray (group A-C) and sand blasting (group D) to four different degrees (roughness average, Ra = group A: 56.1, B: 44.9, C: 28.3, D: 3.7 microns). The interfacial shear strength increased in a surface roughness-dependent manner at both time periods. However, the roughest implants (group A) showed some failed regions in the sprayed layers after pushout test. In the second set of animal experiments (Exp. 2), four kinds of Ti implants; HA-coated smooth Ti (sHA) with Ra of 3.4 microns, bead-coated Ti (Beads), titanium arc sprayed Ti (Ti-spray) with Ra of 38.1 microns and HA-coated Ti-spray (HA + Ti-spray) with Ra of 28.3 microns were compared using the same model as that in Exp. 1. The interfacial shear strength of HA + Ti-spray was significantly greater than that of sHA and Beads at both time periods, and that of Ti-spray at 4 weeks. Although a histological examination revealed that HA-coating enhanced bone ingrowth, sHA showed the lowest shear strength at both time periods. SEM after pushout test showed that sHA consistently demonstrated some regional failure at the HA-implant substrate interface. HA + Ti-spray had many failed regions either at the HA-bone interface or within the bone tissue rather than at the HA-implant substrate interface. These results suggested that the HA-coated smooth surfaced implants had a mechanical weakness at the HA-substrate interface. Therefore, HA should be coated on the rough surfaced implants to avoid a detachment of the HA-coating layer from the substrate and thus obtain a mechanical anchoring strength to bone. HA-coating on this new type of surface morphology may thus lead to a solution to the problems of conventional HA-coated and porous-coated implants.