Theodore R. Oegema

Osteoarthrotic changes after acute transarticular load. An animal model.

The canine patellofemoral joint was subjected to a standardized transarticular load of 2170 newtons for two milliseconds, and the gross and histological changes were examined at two, twelve, and twenty-four weeks after injury. Initially, the load creates fractures in the zone of calcified cartilage, with minimum damage to the articular cartilage surface. Surface fissures were visible in all patellae only after staining with India ink. Histologically, these surface clefts extended into the transitional or superficial radial zone, and they did not communicate with the subchondral bone except in two patellae. However, there were reproducible clefts in the region of the subchondral bone and the zone of calcified cartilage in all patellae. Six months after loading, there was a loss of safranin-O staining above the deep clefts, and there was new-bone formation in the subchondral region and fibrillation of the cartilaginous surface. Thus, the initial changes had progressed to osteoarthrotic-like conditions at six months. In this animal model, the joint is not invaded and the changes that result from loading are reproducible. The injury to the joint creates superficial disruption of the cartilage and subchondral changes that lead to arthritic-like degeneration of the cartilage within six months.

Differential Gene Expression in Ovarian Carcinoma : Identification of Potential Biomarkers

Ovarian cancer remains the fifth leading cause of cancer death for women in the United States. In this study, the gene expression of 20 ovarian carcinomas, 17 ovarian carcinomas metastatic to the omentum, and 50 normal ovaries was determined by Gene Logic Inc. using Affymetrix GeneChip HU_95 arrays containing approximately 12,000 known genes. Differences in gene expression were quantified as fold changes in gene expression in ovarian carcinomas compared to normal ovaries and ovarian carcinoma metastases. Genes up-regulated in ovarian carcinoma tissue samples compared to more than 300 other normal and diseased tissue samples were identified. Seven genes were selected for further screening by immunohistochemistry to determine the presence and localization of the proteins. These seven genes were: the beta8 integrin subunit, bone morphogenetic protein-7, claudin-4, collagen type IX alpha2, cellular retinoic acid binding protein-1, forkhead box J1, and S100 calcium-binding protein A1. Statistical analyses showed that the beta8 integrin subunit, claudin-4, and S100A1 provided the best distinction between ovarian carcinoma and normal ovary tissues, and may serve as the best candidate tumor markers among the seven genes studied. These results suggest that further exploration into other up-regulated genes may identify novel diagnostic, therapeutic, and/or prognostic biomarkers in ovarian carcinoma.

CD44 and β1 Integrin Mediate Ovarian Carcinoma Cell Adhesion to Peritoneal Mesothelial Cells

Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial cells lining the peritoneal cavity. The aim of this study was to identify the adhesion molecules involved in the interaction of ovarian carcinoma cells with mesothelial cells. The human ovarian carcinoma cell lines SKOV3 and NIH:OVCAR5 as well as LP9 cells, a human peritoneal mesothelial cell line, were analyzed by flow cytometry for the expression of CD44 and the β1 integrin subunit. An in vitro adhesion assay was developed whereby LP9 cells were grown as confluent monolayers, and radiolabeled ovarian carcinoma cells were monitored for their ability to adhere to the mesothelial monolayer in the presence of potential inhibitors. Each cell line was evaluated for the presence of a pericellular matrix by a particle exclusion assay. A monoclonal antibody (MAb) against the β1 integrin subunit significantly reduced the adhesion of SKOV3 cells to LP9 cells, whereas NIH:OVCAR5 adhesion to LP9 cells was significantly inhibited by a CD44 MAb. The LP9 cells produced both hyaluronic acid (a ligand for CD44) as well as several extracellular matrix molecules (ligands for the β1 integrin heterodimers). These results suggest that both CD44 and the β1 integrin heterodimers may play a role in mediating the adhesion of ovarian carcinoma cells to mesothelial cells.

Exploiting glycation to stiffen and strengthen tissue-equivalents for tissue engineering

Glycation, the nonenzymatic crosslinking of proteins by reducing sugars, is known to cause stiffening of soft tissues over a lifetime, particularly in diabetics. We show here that glycation due to elevated glucose and ribose concentrations in cell culture medium can be exploited in a matter of a few weeks of incubation to stiffen and strengthen tissue equivalents and to increase their resistance to collagenolytic degradation, all without loss of cell viability. Glycated tissue equivalents did not elicit inflammation or induce calcification upon subcutaneous implantation; rather, they were permissive to host integration and remodeling. Thus a pathological process might be used in a targeted way in tissue engineering to fabricate tissue equivalents with the required mechanical properties and desired resorption rate upon implantation.

The interaction of the zone of calcified cartilage and subchondral bone in osteoarthritis.

The zone of calcified cartilage (ZCC) forms an important interface between cartilage and bone for transmitting force, attaching cartilage to bone, and limiting diffusion from bone to the deeper layers of cartilage. The height of the ZCC is a relatively constant percent of articular cartilage and the height is maintained by a balance between progression of the tidemark into the unmineralized cartilage and changing into bone by vascular invasion and bony remodeling. During its formation, the cells that form the ZCC have properties similar to the cells of the growth plate. In the adult, the ZCC becomes quiescent but not inactive. The ZCC may be reactivated in osteoarthritis and may progressively calcify the unmineralized cartilage. This might contribute to cartilage thinning which would increase the concentration of forces across the uncalcified cartilage leading to more damage.

Heparan Sulfate–Rich Anionic Sites in the Human Glomerular Basement Membrane: Decreased Concentration in Congenital Nephrotic Syndrome

Recent work suggests that the normal barrier to penetration of the renal glomerular basement membrane by anionic plasma proteins may depend in part on the existence of negatively charged sites within the membrane. We describe an in vitro cytochemical method for the quantitative demonstration of anionic sites in the normal human glomerular basement membrane. In five normal subjects, ranging in age from 10 days to 57 years, the sites were distributed at regular intervals in the lamina rara externa, with a frequency of 23.8 +/- 6.8 sites per 1000-nm length of membrane. A similar distribution was observed in the basement membranes from three normal human fetuses. Ex vivo perfusion of one cadaver kidney revealed a similar distribution of anionic sites. The number of anionic sites in the glomerular basement membranes of five patients with the congenital nephrotic syndrome was reduced to 8.9 +/- 3.7 (P less than 0.001). Prior incubation of sections of normal kidney in purified heparinase resulted in a marked reduction in the number of anionic sites. We conclude that congenital nephrosis results from failure of heparan sulfate--rich anionic sites to develop in the lamina rara externa of the glomerular basement membrane.

β1-Integrins Regulate the Formation and Adhesion of Ovarian Carcinoma Multicellular Spheroids

Ovarian carcinoma multicellular spheroids are an in vitro model of micrometastasis whose adhesive abilities have not been elucidated. In this study, we identified adhesion molecules that mediate the formation of ovarian carcinoma spheroids and their subsequent adhesion to extracellular matrix proteins. The NIH:OVCAR5, but not the SKOV3, ovarian carcinoma cell line formed spheroids similar to multicellular aggregates isolated from patient ascitic fluid. NIH:OVCAR5 spheroid formation was augmented by a beta 1-integrin-stimulating monoclonal antibody or exogenous fibronectin, but was inhibited by blocking monoclonal antibodies against the alpha 5- or beta 1-integrin subunits. By immunohistochemical staining, alpha 2-, alpha 3-, alpha 5-, alpha 6-, and beta 1-integrin subunits, CD44, and fibronectin were detected in NIH:OVCAR5 spheroids. NIH:OVCAR5 spheroids adhered to fibronectin, laminin, and type IV collagen, and this adhesion was partially inhibited by blocking antibodies against the alpha 5-, alpha 6-, and alpha 2-integrin subunits, respectively. A blocking monoclonal antibody against the beta 1-integrin subunit completely inhibited adhesion of the spheroids to all three proteins. These results suggest that interactions between the alpha 5 beta 1-integrin and fibronectin mediate the formation of ovarian carcinoma spheroids and that their adhesion to extracellular matrix proteins at sites of secondary tumor growth may be mediated by a complex interaction between multiple integrins and their ligands.

Enhanced fibrin remodeling in vitro with TGF-β1, insulin and plasmin for improved tissue-equivalents

The aim of this study was to better understand how to increase collagen content in and enhance mechanical properties of tissue-equivalents formed by entrapping tissue cells in fibrin gels, with the ultimate goal of developing mechanically robust artificial tissues. The two main areas of focus were cell culture medium composition and replacement frequency relative to a base case of incubating constructs in medium supplemented with just serum and replaced weekly. The optimal condition involved a combination of all three factors investigated-TGF-beta, insulin, plasmin-with medium replacement three times a week. Compared to a base case without these three factors, the combination case resulted in 20 times more collagen and a ten-fold increase in tensile strength. The high strain modulus and tensile strength were within an order of magnitude of cardiovascular tissues. This study indicates great potential for fibrin-based tissue-equivalents in soft connective tissue applications.

Mechanisms of Stiffening and Strengthening in Media-Equivalents Fabricated Using Glycation

We have recently reported that glycation can be exploited to increase the circumferential tensile stiffness and ultimate tensile strength of media-equivalents (MEs) and increase their resistance to collagenolytic degradation, all without loss of cell viability (Girton et al., 1999). The glycated MEs were fabricated by entrapping high passage adult rat aorta SMCs in collagen gel made from pepsin-digested bovine dermal collagen, and incubated for up to 10 weeks in complete medium with 30 mM ribose added. We report here on experiments showing that ME compaction due to traction exerted by the SMCs with consequent alignment of collagen fibrils was necessary to realize the glycation-mediated stiffening and strengthening, but that synthesis of extracellular matrix constituents by these cells likely contributed little, even when 50 micrograms/ml ascorbate was added to the medium. These glycated MEs exhibited a compliance similar to arteries, but possessed less tensile strength and much less burst strength. MEs fabricated with low rather than high passage adult rat aorta SMCs possessed almost ten times greater tensile strength, suggesting that alternative SMCs sources and biopolymer gels may yield sufficient strength by compositional remodeling prior to implantation in addition to the structural remodeling (i.e., circumferential alignment) already obtained.

Manipulation of Hyaluronan Synthase Expression in Prostate Adenocarcinoma Cells Alters Pericellular Matrix Retention and Adhesion to Bone Marrow Endothelial Cells

Prostate cancer metastasis to bone marrow involves initial adhesion of tumor cells to the bone marrow endothelium, followed by transmigration and proliferation within the marrow. Rapid, specific adhesion of highly metastatic prostate adenocarcinoma cells (PC3M-LN4) to bone marrow endothelial cell (BMEC) lines requires a pericellular hyaluronan (HA) matrix and correlates with dramatically up-regulated HA synthase (HAS) expression. Non-metastatic prostate tumor cells (LNCaP) do not assemble a HA matrix, adhere poorly to BMECs, and express normal levels of HAS. Preferential bone metastasis of prostate carcinoma cells may therefore be facilitated by tumor cell HA biosynthesis. In this report, HAS gene expression was manipulated to investigate the direct impact of prostate tumor cell HA production on adhesion to BMECs. PC3M-LN4 cells stably transfected with antisense HAS2 and HAS3 failed to form pericellular matrices. Adhesion of these transfectants to BMECs was significantly diminished, comparable to the low level exhibited by LNCaP cells. Upon transfection with full-length HAS2 or HAS3, the non-adherent LNCaP cells retained pericellular HA and adhered to BMECs. The results of this study are consistent with a model in which HA matrix formation, BMEC adhesion, and metastatic potential are mediated by HAS expression.