Molecular characterization of gene expression changes in ROS 17/2.8 cells cultured in diffusion chambers in vivo.

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3-10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3-28 days and by 56 days was 4-5 fold above control levels. In 3-10 days, c-jun expression increased 1.6-1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10-56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to

Hepatocellular proliferation in ibuprofen-treated mice.

Female B6C3F1 mice were treated with ibuprofen for 2 wk or 90 days to monitor effects on hepatocellular proliferation during acute and subchronic exposure. Proliferation was assessed by bromodeoxyuridine labeling. Mice treated with 100, 200, or 400 mg/kg ibuprofen for 2 wk had significantly increased liver weights. A dose-related increase in the number of labeled hepatocytes per 1,000 hepatocytes suggested that the weight increases were in part a result of hepatocellular proliferation. Hepatocellular hypertrophy also contributed to the increased liver size as indicated by decreases in the number of hepatocytes per high power field. Ultrastructural evaluation indicated that hepatocyte peroxisome size increased significantly in treated mice. Mice treated with 100 or 200 mg/kg ibuprofen for 90 days and given bromodeoxyuridine during the last 2 wk of ibuprofen exposure had statistically significant increases in relative liver weights. However, the number of labeled hepatocytes per 1,000 hepatocytes was not increased, and there was no evidence of hepatocellular hypertrophy. Mice given 200 mg/kg ibuprofen for 90 days had significantly decreased serum triglycerides. These findings indicate that ibuprofen treatment of mice results in hepatomegaly characterized by hepatocellular hypertrophy and hyperplasia. Peroxisomal changes may be contributory to the pathogenesis of this lesion.

Effects of body weight restriction on the development and progression of spontaneous osteoarthritis in guinea pigs.

Hartley albino guinea pigs develop spontaneous osteoarthritis (OA) of the knee joint. A study was done to determine the importance of body weight in the pathogenesis of this disease. Two groups of 20 male guinea pigs each were maintained on the same diets. The control group was allowed ad libitum feed consumption and the other group was restricted to 30-35 gm of feed per day. Ten animals from each group were killed at 9 months of age to evaluate histologic features of the knee joints. The severity of the OA lesions was reduced by 40%, in conjunction with a 28% decrease in body weight, in the diet-restricted group. The remaining animals were killed at 18 months of age. Those in the diet-restricted group had a 56% reduction in severity of lesions, with a 29% decrease in body weight. These results indicate that body mass in guinea pigs, as in humans, is an important predisposing factor for the development of spontaneous OA of the knee.

Passive role of articular chondrocytes in the pathogenesis of acute meniscectomy-induced cartilage degeneration.

The importance of viable articular chondrocytes and enzymes or factors from surgically traumatized synovium in the pathogenesis of acute meniscectomy-induced cartilage degeneration was examined in guinea pigs (nine groups of five animals each). Iodoacetate injected intra-articularly was used to kill articular chondrocytes of guinea pigs prior to meniscectomy to determine if they were active participants in the acute lesion induction. Lesions of similar severity to those occurring in animals with viable chondrocytes at the time of surgery were observed, suggesting that the chondrocytes were not actively involved in the pathogenesis in this group. In an additional group of guinea pigs in which chondrocytes were killed by iodoacetate, the medial collateral ligament was transected to determine if acute degenerative changes could be induced in acellular cartilage exposed to a surgical manipulation that does not by itself induce lesions but does expose cartilage to enzymes/factors from traumatized synovium. Transecting the medial collateral ligament and entering the joint space without induction of instability via meniscectomy did not result in histologic evidence of cartilage damage. This suggests that synovial trauma and mild inflammation were insufficient to induce matrix degeneration in the absence of abnormal load bearing. In further support of this, guinea pigs subjected to unilateral sciatic neurectomy at the time of meniscectomy were protected against development of acute cartilage degeneration. Results of this study suggest that articular cartilage devoid of viable chondrocytes at the time of meniscectomy responds acutely in much the same way as intact cartilage subjected to this procedure.

Passive role of articular chondrocytes in quinolone-induced arthropathy in guinea pigs.

The role of articular chondrocytes and matrix degrading enzymes such as collagenase and neutral protease in the pathogenesis of quinolone-induced cartilage degeneration was investigated in immature guinea pigs. Articular cartilage from nalidixic acid (NA) treated guinea pigs was examined for the presence of protease activity or the ex vivo synthesis of collagenase at various times post-treatment. Histologic evaluation of knee joints confirmed the presence of degenerative changes in the matrix, but increased collagenase synthesis or protease activity were not detected. A separate group of animals was used to determine the importance of articular chondrocytes in the lesion generation. These cells were killed by intra-articular injection of the glycolysis inhibitor iodoacetic acid (IA) prior to treatment of the animals with NA. Typical "blister-like" their presence was not required for lesion development. Cartilage exposed to IA only did not exhibit "blister-like" lesions indicating that chondrocyte death and proteoglycan loss in conjunction with frictional forces associated with load-bearing were not sufficient to induce major matrical disruptive changes during the period of this study. These results indicate that articular chondrocytes are not actively involved in inducing the degenerative changes and provide no evidence for involvement of collagenase or neutral protease in the pathogenesis of the lesion.

Osteoarthrosis in guinea pigs: histopathologic and scanning electron microscopic features.

Spontaneous cartilage degeneration of the femorotibial joint of male Hartley guinea pigs, 61 to 365 days old, was studied by light microscopy (LM) and scanning electron microscopy (SEM) to determine the incidence, age at onset, and to characterize the early changes. Knee joints of 61 day old animals were histologically and ultrastructurally normal. Focal minimal degeneration characterized by cell and proteoglycan loss with surface fibrillation was first observed by LM on the medial tibial plateau (MTP) in two of five 89 day old animals. Mild lesions characterized by focal surface disruption, primarily in the area of medial tibial plateau not covered by the meniscus, were observed in three of five 89 day old animals by SEM. Light microscopic alterations in knee joints of 4, 5, and 6 month old animals consisted of varying degrees of focal chondrocyte death, decreased toluidine blue matrix staining, and surface fibrillation. Small chondrocytic clones were first observed in medial tibial cartilage of 6 month old animals with moderate focal degeneration. Ultrastructurally, 4, 5, and 6 month old animals generally had moderate to severe fibrillation involving primarily the area of the medial tibial plateau not covered by the meniscus. Tibial osteophyte formation, mild synovial hyperplasia, medial femoral and meniscal cartilage degeneration, were first seen by LM in 9 month old animals. Lesions in 1 year old animals were similar, but more severe and included subchondral sclerosis of medial tibial and femoral bone. Bilateral fibrillation of greater than 50% of the medial tibial articular surface was observed in all 1 year old animals by SEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous cartilage degeneration in guinea pigs.

Spontaneous degeneration of the knee joint cartilage in male Hartley guinea pigs was studied by light microscopy in animals aged 3-18 months. Unilateral focal degeneration, characterized by chondrocyte death and proteoglycan loss with surface fibrillation, was observed on the medial tibial plateau in 2 of 5 guinea pigs that were 3 months old. The incidence and severity of the lesions increased with age, and by 12-18 months of age, all animals had moderate-to-severe degeneration of the medial tibial plateau, femoral condyle, and meniscus. Lesions were not present on the lateral aspect of the knee joint in any of the animals.