Predictive value of serum immunosuppressive acidic protein for lung metastasis after amputation of murine osteosarcoma of the lower limb.

A spontaneously occurring murine osteosarcoma cell line (POS-1) was inoculated into the footpads of 88 mice. In 59 mice, the tumor-bearing leg was amputated at 1, 3, 5, or 7 weeks after inoculation and the mice sacrificed at 9 weeks. Lung metastasis was observed in 82.6-100% of the mice undergoing leg amputation at 5 or 7 weeks after inoculation. There were no lung metastasis in the mice amputated at 1 or 3 weeks. The serum immunosuppressive acidic protein (IAP) concentration showed a significant increase at 5 or 7 weeks after inoculation (P<0.005 and P<0.0005, respectively). When amputation was done 5 weeks after inoculation, the serum IAP concentration increased further to 5.9 times the pre-amputation level 1 week after the amputation. The IAP concentration increased only 1.3-fold in the control group without inoculation. Thus, a high serum IAP concentration before amputation and a further increase after amputation were indicators of lung metastasis in this mouse model of osteosarcoma.

Augmentation of anti-tumor effects of methotrexate by distilled water on Dunn osteosarcoma in mouse air pouch.

The anti-tumor effects of hypoosmotic solution of MTX in distilled water (DW) on Dunn osteosarcoma were evaluated in mouse air pouches. Dunn osteosarcoma cell suspension (1 x 10[5] cells in 0.1 ml of medium) was inoculated into the mouse subcutaneous air pouch that had formed 7 days after the initial injection of air. Two hours after the inoculation of tumor cells, 5 ml of various concentrations of MTX (from 0 to 1 x 10[-3] M) dissolved in DW or PBS were injected into the air pouch. Five minutes later, the entire solution in the air pouch was aspirated. The mice were sacrificed 3 weeks after the inoculation of tumor cells and the air-pouch tissue was transected in the coronal plane with the largest area of tumor mass. The sections were stained with H&E and the area was measured with the NIH Image program. The largest area of tumor mass in the air pouch treated with 1 x 10(-3) M of MTX in DW was 11.8+/-3.4 mm2 (N = 5), which was significantly (P < 0.005) smaller than that in PBS (51.7+/-8.3 mm2). These findings suggested that hypoosmotic solution in DW might augment the anti-tumor effect of MTX on sarcoma cells.

Acceleration of lung metastasis by up-regulation of CD44 expression in osteosarcoma-derived cell transplanted mice.

The effect of CD44-phenotypic expression on metastasis to the lung was studied using a spontaneous murine osteosarcoma-derived cell line, POS-1, stimulated with lipopolysaccharide (LPS). POS-1 cells were inoculated into the hind paws of 20 C3H/HeJ mice and produced a visible mass in all mice in 5 weeks, and these transplanted tumors resulted in lung metastasis in all mice. The number of metastatic foci in the lungs was 12.0+/-2.1 (mean+/-SD) with LPS-stimulated cells, which was significantly higher than that of unstimulated cells (5.8+/-1.4; N=10 for each; P<0.05). Hyaluronate (HA), a ligand of CD44, inhibited a number of lung metastases in a dose-dependent manner (0.5% HA, 3.0+/-1.1; 0.005% HA, 5.1+/-1.5; without HA, 8.6+/-1.7; N=10 for each; P<0.05, each group with HA versus the group without HA). Adhesion assay by coculturing POS-1 cells and lung microvascular endothelial cells on culture plate showed that the adhesion was significantly lower in HA treated POS-1 than those without HA (1.18+/-0.12 and 2.74+/-0.17, respectively, P<0.05). These results suggest that lung metastasis was accelerated by up-regulation of CD44.

Highly activated matrix metalloproteinase-2 secreted from clones of metastatic lung nodules of nude mice injected with human fibrosarcoma HT1080.

The promoting effects of matrix metalloproteinase-2 (MMP-2) on lung metastasis of human fibrosarcoma cells (HT1080) were studied using nude mice. The fourth generation of HT1080 was established by consecutive clonal selection of metastatic lung nodules formed by intravenous transplantation. MMP-2 and MMP-9 in the culture supernatants of the first and fourth generation cells were analyzed by gelatin zymography and Western blotting, and quantified by scanning densitometry. In gelatin zymograms, mean ratios of values for the 59-kDa band (the active form of MMP-2) to those for the 72-kDa band (the inactive form of MMP-2) for optical density; area, and volume measured by densitometry were 1.44 +/- 0.12, 0.93 +/- 0.05, and 1.27 +/- 0.20, respectively, in the culture supernatant of fourth generation cells isolated from metastatic lung nodules. These values were significantly (P < 0.01) higher than those of first generation cells (0.70 +/- 0.04, 0.48 +/- 0.01, and 0.57 +/- 0.42). Three weeks after intravenous transplantation of HT1080 cells into nude mice, the incidence of lung metastasis and mean number and diameter of metastatic nodules formed by injection of first generation cells were 20% (2 of 10 mice), 2.9 +/- 0.2 and 2.0 +/- 0.2 mm, respectively; while they were 100%, 99.8 +/- 7.2 and 4.3 +/- 0.3 mm following injection of fourth generation cells. These findings suggest that the active MMP-2 produced by human fibrosarcoma cells is important for the cells to form lung metastatic lesions in nude mice.

Effects of lipo prostaglandin E1 on distribution of cis-diamminedichloroplatinum in lung metastasis derived from Dunn osteosarcoma cell-inoculated mouse foot-pad.

This study was designed to determine the effect of lipid microspheres containing prostaglandin E1 (lipo PGE1) on cis-diamminedichloroplatinum (CDDP) accumulation in primary and lung metastatic lesions. Sixty mice were divided into four groups, depending on whether or not an intra-foot-pad injection of Dunn osteosarcoma cells had been administered and on whether or not an intraperitoneal injection of lipo PGE1 had been administered. CDDP was injected intraperitoneally into all the mice 6 weeks after the inoculation. Tumor colonies of spontaneous metastases in the left lung were found in 21 out of 30 tumor-inoculated mice at autopsy. Tissue platinum concentrations in the lungs with metastatic colonies and in the foot-pad tumors were significantly higher in the lipo PGE1-administered mice than in those without treatment. Terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) assay showed marked localization of dying cells in the lung metastatic lesions of the lipo PGE1-administered mice. The results of this study showed that pretreatment with lipo PGE1 may augment the antitumor effects of CDDP at the tumor site.

Does the Adamkiewicz artery originate from the larger segmental arteries?

OBJECTIVE: The Adamkiewicz artery supplies most of the blood to the anterior spinal artery, which perfuses the anterior two thirds of the spinal cord. During operations for thoracoabdominal aortic aneurysm, detailed anatomic knowledge of the Adamkiewicz artery and its correlation with the intercostal and/or lumbar arteries is important to prevent postoperative paraplegia. METHODS: Minute dissection was performed on 102 formol-fixed adult cadavers without any history of circulatory disorders. The Adamkiewicz artery was found in the epidural space after laminectomy of the vertebrae. The entire course between the Adamkiewicz artery and the intercostal and/or lumbar artery was dissected carefully. The vertebral level, laterality, and mean diameter of all Adamkiewicz arteries were investigated. The correlation between the diameter of the Adamkiewicz artery and that of the intercostal and/or lumbar arteries was also determined. RESULTS: The mean number of Adamkiewicz arteries per cadaver was 1.3 +/- 0.65, and the mean diameter was 0.77 +/- 0.24 mm (range, 0.50 to 1.49 mm). Approximately 70% of the Adamkiewicz arteries originated from the intercostal and/or lumbar arteries on the left side, frequently at the T8-L1 vertebral level. There was no statistically significant correlation between the diameter of the Adamkiewicz artery and that of intercostal and/or lumbar arteries. CONCLUSION: This study provides evidence that, during operations on the thoracoabdominal aorta, the intercostal and/or lumbar arteries should be preserved, regardless of their diameter, to prevent postoperative paraplegia.

OP-1206, a prostaglandin E1 derivative. Effects of oral administration to patients with chronic lung disease.

In order to assess the clinical effectiveness of an oral prostaglandin E1 derivative, OP-1206, five patients with chronic obstructive pulmonary disease and two with pulmonary fibrosis were studied from the standpoint of hemodynamics and nine others with chronic lung disease from the standpoint of respiratory function. Oral intake of OP-1206 resulted in a significant decrease in the pulmonary arterial pressure (p less than 0.01), total pulmonary vascular resistance (p less than 0.01), pulmonary arteriole resistance (p less than 0.01), and total systemic vascular resistance (p less than 0.05), and an increase in the cardiac index (p less than 0.05) and oxygen delivery (p less than 0.01) with insignificant changes of PaCO2, PaO2 and pH. There was no clinical improvement of lung function after OP-1206 intake. OP-1206 is a potent vasodilator, improving cardiac performance in patients with chronic lung disease and possibly preventing the progress of cor pulmonale in this kind of patient.

Compressive and bone-bonding strength of hydroxyapatite thermal decomposition product implanted in the femur of rabbit as a bioactive ceramic bone cement.

Hydroxyapatite thermal decomposition product (HATDP) after mixing with water was injected into the femur of rabbits, and its biocompatibility, compressive strength and bone-bonding strength were investigated. The compressive strength of a block of HATDP was 56.0 MPa 4 weeks after injection, which was about 60% of that of poly(methyl methacrylate) (PMMA) cement. The bone-bonding strength was 8.2 MPa 4 weeks after injection, which was significantly higher than that of PMMA cement. HATDP seems to be useful as a cement material because of its good bone-bonding property and relatively high mechanical strength.

Bone formation as a reaction to hydraulic hydroxyapatite thermal decomposition product used as bone cement in rabbits.

The bone activity of hydroxyapatite thermal decomposition product was evaluated by the amount of new bone formation after implantation into the femur of rabbits. New bone formation was observed to be bonded to the entire adjacent surface of implanted ceramics at the metaphysis in 16 out of 20 specimens 12 weeks after implantation.

Viscous property and osteogenesis induction of hydroxyapatite thermal decomposition product mixed with gelatin implanted into rabbit femurs.

The viscosity of hydroxyapatite thermal decomposition product mixed with gelatin (HATDP-G) and the osteogenesis induced by this mixture were investigated. To determine the viscosity of HATDP-G, we measured the average diameter of a slumped mass of HATDP-G (0.5 ml) according to the American Dental Association Specification No. 8, with some modifications, and compared it with that of an equal mass of HATDP and of polymethylmethacrylate (PMMA) bone cement. The mean diameter of 10 samples each of HATDP-G, HATDP, and PMMA bone cement was 26.8+/-0.9 mm, 19.6+/-0.2 mm and 29.2+/-0.3 mm, respectively, 2 min after the mixing procedure. HATDP-G was injected into the bone marrow of the right femurs of 25 rabbits. As the control, gelatin with saline solution was injected into the left femurs. New bone formation was observed in all 20 femurs from three weeks after injection. No new bone formation was observed in the control femurs. The affinity index, a parameter of osteogenesis, was 19.8+/-6.1%, 27.5+/-2.6%, 52.9+/-9.5%, and 76.3+/-1.9% at 3, 6, 12 and 24 weeks after the injection, respectively. Significant increases in the affinity index were found between weeks 6 and 12, and weeks 12 and 24 (p < 0.05). Our findings indicated that HATDP-G might be successfully used as a bioactive bone cement.

Osteogenic response of rabbit tibia to hydroxyapatite particle-Plaster of Paris mixture.

A mixture of hydroxyapatite (HA) particles and plaster of Paris (POP) with saline solution was injected into the bone marrow of 30 unilateral tibiae of 30 rabbits. As a control, POP with saline solution was similarly injected into the bone marrow of 30 unilateral tibiae of 30 additional rabbits. Five rabbits in each group were killed 2, 4, 8, 12, 24 and 40 weeks after injection. Histological examination was performed on three tibiae of five killed animals, and X-ray diffraction analysis of the bone marrow was performed on the remaining two tibiae. In two of three tibiae, bridges of new bone were formed between the hydroxyapatite particles and the cortical bone, 4 weeks after the injection of the HA particle-POP mixture. New bone formation was seen in all three tibiae at 8 weeks and thereafter. No new bone formation was observed in the tibiae of the control group. The affinity index, a parameter of osteogenesis, was 0% at 2 weeks, 52.2+/-14.0% at 4 weeks, 74.8+/-5.3% at 8 weeks, 86.1+/-6.1% at 12 weeks, 90.1+/-8.3% at 24 weeks and 92.8+/-4.1% at 40 weeks after injection. Statistically significant increases were found between the affinity indexes of 2 weeks and 4 weeks and between those of 4 weeks and 8 weeks, p < 0.03 and p < 0.05, respectively. Between 12, 24 and 40 weeks, the indexes showed tendencies toward increases. X-ray diffraction of bone marrow of two tibiae into which HA particle-POP mixture or POP alone was injected showed that POP had disappeared 8 weeks after injection.

Osteogenic response of the rabbit femur to a hydroxyapatite thermal decomposition product-fibrin glue mixture.

The effect of fibrin glue on the hardening process of hydroxyapatite thermal decomposition product (HATDP) was investigated in vitro. To study, in vivo, the effect of fibrin glue on osteogenesis, a mixture of HATDP with fibrin glue was injected into the left femurs of 20 rabbits, and HATDP without fibrin glue was injected into the right femurs. New bone formation was observed in the area of direct contact with the fibrin-containing mixture in the metaphysis at 2 weeks after injection in two of four femurs, but not in the fibrin-free group. At 4 weeks after injection, new bone formed a bridge between the mixture and the cortical bone in three of the four specimens. A parameter of osteogenesis (the affinity index) was calculated at 4, 12 and 24 weeks after injection. It was respectively 0.14+/-0.02, 0.49+/-0.12 and 0.44+/-0.05 for the fibrin-containing mixture, and 0.14+/-0.062, 0.44+/-0.15 and 0.49+/-0.04 for the control. A comparable but significant increase in the average affinity index was noted over 12 weeks in both the fibrin-containing and the fibrin-free HATDP. These findings indicate that fibrin glue accelerated the hardening of HATDP but did not inhibit the process of osteogenesis.

Osseous tissue reaction around hydroxyapatite block implanted into proximal metaphysis of tibia of rat with collagen-induced arthritis.

This study was conducted to investigate the influence of osteoporosis on new bone formation around a hydroxyapatite (HA) block implanted into the proximal metaphysis of the tibia of rats with collagen-induced arthritis (CIA). Ten rats were immunized with an emulsion of bovine type II collagen and Freund's complete adjuvant (arthritis group). Another 10 rats, which were not immunized were used as the control group. Seventeen days after immunization, HA block was implanted into the proximal metaphysis of the tibia. Four weeks after implantation, all rats were killed. The serum level of tetrate-resistant acid phosphatase (TRAP), bone mineral density (BMD) in the proximal metaphysis of the tibia and the affinity index in the arthritis group were 28.0+/-3.5 IU/ml, 130.3+/-28.7 mg/cm2 and 77.6+/-10.8%, respectively, and those in the control group were 24.6+/-5.5 IU/ml, 175.9+/-30.5 mg/cm2 and 56.3+/-14.8%. The serum level of TRAP was higher (P < 0.05) and BMD was lower (P < 0.005) in the arthritis group. The amount of new bone formation around the HA block was larger (affinity index, P < 0.05) in the arthritis group than in the control group. These findings suggest that bone formation around HA block might be enhanced even in conditions associated with highly activated bone resorption and bone formation, such as arthritis.

New bone formation around porous hydroxyapatite wedge implanted in opening wedge high tibial osteotomy in patients with osteoarthritis.

A porous hydroxyapatite (highly purified synthetic Ca10(PO4)6(OH)2) wedge was inserted into the tibia in 10 knees of 7 patients with osteoarthritis of the knee who underwent high tibial osteotomy. The interface of this HA wedge with bone was histologically examined in undecalcified specimens obtained at the time of hardware removal in all 10 knees, and total incorporation of the HA into bone was observed, with no inflammatory reaction. The proportion of pores that were filled with regenerated bone within 300 microm from the interface was 71.8 +/- 10.1% (mean +/- S.D.), and it was positively correlated with the time from implantation to biopsy. Radiodensity of the HA wedge was measured during the follow-up period, and it did not change significantly within 36 months after osteotomy. Our study concluded that bone ingrowth into the HA block slowly progressed and was correlated with the passage of time.

Effects of collagen gel mixed with hydroxyapatite powder on interface between newly formed bone and grafted achilles tendon in rabbit femoral bone tunnel.

The Achilles tendon was implanted into a bone tunnel made in the femoral condyle of 20 rabbits. In the left femur, collagen gel mixed with hydroxyapatite powder (C-HAp) was injected between the graft and the bone tunnel. On the other hand, as a control, simple saline was injected in the right femur. Five rabbits were sacrificed at 4, 8, 12, and 16 weeks after surgery. Histological findings showed that in the C-HAp group, the grafted tendon came in direct contact with new bone, and Sharpey-like collagen fibers arising from the grafted tendon were observed to penetrate new bone by 4 weeks after surgery. In the control group, however, fibrous tissue was observed between new bone and the grafted tendon, but no penetrating fibers from the grafted tendon into the new bone were observed until 16 weeks. The area of new bone in the C-HAp group was significantly greater than that in the control group 4, 8, and 12 weeks after surgery (p < 0.0001, p < 0.0007, p < 0.0013, respectively).

Successful surgical treatment of giant coronary artery aneurysm with fistula.

Giant coronary artery aneurysm with fistula formation is a rare entity. We report a giant coronary artery aneurysm with a maximum diameter of 70 mm with fistula, in which a favorable course was obtained after surgical treatment. We also review the literature on giant coronary artery aneurysms exceeding 50 mm in maximum diameter.

Enlargement of ulcer-like projections after repair of acute type A aortic dissection.

We treated two cases of enlargement of ulcer-like projections in the descending thoracic aorta, which were recognized after emergency graft replacement from the ascending aorta to the aortic arch for acute type A aortic dissection. The intimal tear, which was near the left subclavian artery, was resected during the initial operation. Graft replacement of the descending thoracic aorta was successful.

Effect of the immunosuppressant drug FK506 on neonatal cerebral mitochondrial function and energy metabolism after transient intrauterine ischemia in rats.

Mitochondrial respiratory activities and energy metabolism were measured in neonatal rat brains to evaluate the influence of transient intrauterine ischemia on the near-term fetus and to assess the effect of the immunosuppressant drug FK506 treatment. Transient intrauterine ischemia was induced by 30 min of right uterine artery occlusion at 17 days of gestation in Wistar rats. The vehicle or 1.0 mg/kg of FK506 was administered after 1 h of recirculation. All of the pups were delivered by cesarean section at 21 days of gestation and samples of cerebral cortical tissue were obtained from pups at 1 h after birth. The mitochondrial respiration was measured polarographically in homogenates. For the analysis of ATP, ADP, and AMP, neonatal brains were frozen in situ and fluorometric enzymatic techniques were used. In the neonatal cortical tissue exposed to ischemia, mitochondrial respiratory activities and ATP concentrations decreased significantly to approximately 59 and 67% of those in normoxic controls, respectively. The deterioration of both mitochondrial respiratory activities and high-energy phosphates was prevented by FK506, given 1 h after the start of recirculation. The present results indicate that transient intrauterine ischemia is accompanied by mitochondrial dysfunction and cellular bioenergetic failure in the neonatal rat brain and suggest that treatment with FK506 prevents the deterioration, even when administered after the ischemic periods.

Comparison of cyclooxygenase-1 and -2 inhibitory activities of various nonsteroidal anti-inflammatory drugs using human platelets and synovial cells.

Recent studies have shown that cyclooxygenase exists in two isozyme forms. Since differences in the pharmacological profiles of nonsteroidal anti-inflammatory drugs (NSAIDs) might be accounted for by varying degrees of selectivity for these isozymes, cyclooxygenase-1 and -2, the relative potency of various NSAIDs in inhibiting their activities was examined in intact human cells. We used human platelets cyclooxygenase-1 and interleukin-1beta-stimulated human synovial cell cyclooxygenase-2 for measuring cyclooxygenase selectivity. The presence of the enzymes was confirmed by immunoblotting and immunoprecipitation analysis, and by the reverse transcriptase-polymerase chain reaction. Mean IC50 values (microM) for human platelet cyclooxygenase-1 and interleukin-1beta-stimulated human synovial cell cyclooxygenase-2 and cyclooxygenase-1/-2 IC50 ratio of various NSAIDs were as follows: aspirin, 3.2, 26, 0.12; diclofenac, 0.037, 0.00097, 38; etodolac, 122, 0.68, 179; ibuprofen, 3.0, 3.5, 0.86; indomethacin, 0.013, 0.044, 0.30; loxoprofen (active metabolite), 0.38, 0.12, 3.2; NS-398, 12, 0.0095, 1263; oxaprozin, 2.2, 36, 0.061; zaltoprofen, 1.3, 0.34, 3.8; respectively. Our bioassay system employing intact human cells to assess the cyclooxygenase selectivity of NSAIDs may provide clinically useful information.

Aceclofenac blocks prostaglandin E2 production following its intracellular conversion into cyclooxygenase inhibitors.

Aceclofenac, 2-[(2,6-dichlorophenyl) amino] phenylacetoxyacetic acid, is a novel non-steroidal anti-inflammatory drug. We investigated the effects of aceclofenac on prostaglandin E2 production by several kinds of human cells. Aceclofenac inhibited interleukin-1beta-induced prostaglandin E2 production by human rheumatoid synovial cells, but had no inhibitory effect on cyclooxygenase-1 or cyclooxygenase-2 activities by itself. We also observed that part of the aceclofenac was converted into diclofenac, the cyclooxygenase-1 and cyclooxygenase-2 inhibitor, when aceclofenac was incubated with human rheumatoid synovial cells. Aceclofenac was also converted into diclofenac and 4'-hydroxy diclofenac by human polymorphonuclear leukocytes and monocytes. 4'-Hydroxy diclofenac suppressed prostaglandin E2 production specifically by blocking cyclooxygenase-2 activity. These findings suggested that aceclofenac can be metabolized to cyclooxygenase inhibitors (diclofenac and/or 4'-hydroxy diclofenac) by these inflammatory cells. Although detailed examinations in non-inflammatory cells remain to be studied, we concluded that aceclofenac is shown to be a new type of non-steroidal anti-inflammatory drug which is intracellulary converted into active metabolites that inhibit the prostaglandin E2 production.