The protective effect of the blood brain barrier from systemic cytokines in an animal femur fracture model.

BACKGROUND: Previous studies of head trauma have shown profound release of cytokines in the brain. These changes were not expressed in peripheral tissues. The intent of this study was to take an animal model of femur fracture, monitor the expression of biochemical markers in the periphery, and compare this to their expression in the brain. METHODS: Rats were subjected to a weight-drop, femur fracture model, and then killed at various times. Samples of muscle, liver, serum, and brain were analyzed for concentrations of cytokines, and compared with controls. RESULTS: Statistically significant (p < 0.05) results from the study were found in the liver. Interleukin (IL)-2, IL-10, IL-11, and other acute phase reactants were elevated at 24 hours after injury, compared with in controls. Analysis of these cytokines in the brain showed no significant increase when compared with those of controls. Further analysis also demonstrated an increase in plasma C-reactive protein and leptin in the fracture group. These results differ from our previous brain trauma study, which demonstrated no increased expression of cytokines in liver or plasma. CONCLUSIONS: This animal model of peripheral injury demonstrates that there is a significant rise in acute phase reactants in liver tissue and plasma within 24 hours after injury, without a corresponding rise in cytokine concentration in the brain. These results suggest that although the brain is potentially exposed to the biochemical response to injury, the brain parenchyma itself is protected from up-regulation of proinflammatory cytokines. Interestingly, this is the opposite effect seen in our isolated brain injury study.

Isolation of large numbers of mesenchymal stem cells from the washings of bone marrow collection bags: characterization of fresh mesenchymal stem cells.

BACKGROUND: Bone marrow (BM) is an excellent source of mesenchymal stem cells (MSC) which can be expanded in vitro for further use. However, large volumes of BM specimens are not routinely available. We hypothesized that the normally discarded BM collection kits might be a convenient source of large numbers of MSC. METHODS: Marrow specimens were isolated from used Fenwal collection kits. Purified mononuclear cells (MNC) were screened by multiparameter flow cytometry to identify MSC, which were later expanded by in vitro culture. Immunophenotyping and differentiation assays were performed initially and at subculture. Both fresh and frozen BM were tested. RESULTS: An average of 9.62E+08 MNC were collected. In this, a cell population was identified that was CD44+, CD73+, CD90+ and CD105+, but negative for hematopoietic markers. This population represented on average 0.015% of the total BM MNC fraction, or on average 1 in 6,666 MNC. The population was considered to be MSC based on its immunophenotype profile, suppressive ability in mixed lymphocyte cultures, morphology, and ability to differentiate into bone and fat cells. CONCLUSIONS: This study demonstrates that large numbers of MSC can be obtained from the normally discarded collection devices following harvest of BM for clinical transplant. This novel method offers potential for obtaining large numbers of MSC for potential therapeutic or investigational purposes following their in vitro expansion.

Hyaluronic acid of high molecular weight inhibits proliferation and induces cell death in U937 macrophage cells.

Hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix, has regulatory influences on cells and cellular activities. To explore the effects of a high concentration (1 mg/mL) of high molecular weight HA (500-730 kD) on U937 macrophage growth dynamics, three factors that influence overall cellular growth, namely proliferation, apoptosis, and cell death, were examined. Cells were cultured with HA and were analyzed by flow cytometry every 24 hours during a 168-hour period for proliferation and the presence of apoptotic and dead cells. These analyses demonstrated that HA inhibits U937 macrophage proliferation in a time-dependent manner. Through the first 72 hours, cells exhibited slowed proliferation. However, no evidence of cell division arrest or reduced cell viability was observed. Thereafter, HA continued to diminish proliferation, but induced apoptosis. This data is consistent with regulatory influences secondary to HA binding to CD44 and/or RHAMM cell surface receptors, both of which were shown to be expressed on U937 macrophages. This study demonstrates that a high concentration of high molecular weight HA greatly inhibits macrophage population growth by the dual actions of impeding cell proliferation and inducing apoptosis.

Hyalgan has a dose-dependent differential effect on macrophage proliferation and cell death.

The intra-articular injection of high molecular weight hyaluronic acid (HA) has been reported to be an effective treatment for pain of osteoarthritis of the knee. However, the mechanism by which HA exerts its effect is unknown. To explore HA's influence on the growth of U937 human macrophages, cells were incubated for 168 h with three concentrations, 1, 0.1 and 0.01 mg/mL, of Hyalgan, a high molecular weight HA preparation. At 24-h increments, the cells were examined for proliferation, cell cycle distribution as well as the number of apoptotic and dead cells. Exposing macrophages to 1 mg/mL Hyalgan significantly reduced the rate of cellular proliferation and altered the cell cycle distribution to yield decreased proportions of G0/G1 cells but increased S and G2/M cells. Concomitantly, a 10-fold increase in apoptotic cells and a 12-fold increase in dead cells were observed. The population doubling time (PDT) for cells treated with 1.0 mg/mL Hyalgan increased from 23.6 to 52.9 h. By contrast, the two lower Hyalgan concentrations significantly promoted macrophage proliferation in a dose-dependent manner. They also increased the proportion of G2/M cells, but had no effect on the number of apoptotic or dead cells. The PDTs of 21.5 and 22.2 h were less than the control time of 23.6 h. These results demonstrate that Hyalgan concentrations have a differential effect on macrophage growth dynamics and suggest an anti-inflammatory effect at high HA concentrations.

Long-term survival of McKee-Farrar total hip prostheses.

Because of the recent resurgence of interest in metal-on-metal bearing components for total hip arthroplasties, the long-term results of 153 consecutive McKee-Farrar total hip arthroplasties done in 129 patients by one surgeon between 1969 and 1973 were evaluated. A retrospective chart review provided patient demographics (age, gender, weight, primary diagnosis), revision dates, indications, and implant survival data. The average age of the patients at implantation surgery was 61 years (range, 28-85 years) and these patients were observed as many as 28 years. Primary diagnoses included osteoarthritis (49% of implants), rheumatoid arthritis (38%), and other conditions (13%). During the 28 years of followup, five implants were revised for infection and 14 implants were revised for aseptic loosening. Survivorship analysis of the McKee-Farrar prostheses had a 20-year probability of implant survivorship of 84%, and a 28-year implant survivorship of 74%. Excellent long-term results of the McKee-Farrar prosthesis were seen. Given the inherent problems associated with implant wear debris, especially polyethylene wear particles, second generation metal-on-metal bearing implants may offer a viable alternative to current designs. Their excellent long-term survival may infer particular suitability for use in younger patients.

Macrophage exposure to particulate titanium induces phosphorylation of the protein tyrosine kinase lyn and the phospholipases Cgamma-1 and Cgamma-2.

A frequent long-term complication of total joint arthroplasty is aseptic loosening, the end result of wear debris production, synovial macrophage activation, inflammatory mediator release, and osteolysis about the implant-bone or cement-bone interface. To elucidate the mechanisms of particle-induced macrophage activation and mediator production, we studied early signal transduction events using J774A.1 macrophages and 3 microm titanium particles. Treating macrophages with herbimycin A or genistein, two inhibitors of protein tyrosine kinases (PTKs), inhibited titanium phagocytosis as well as secretion of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin-E2 (PGE2) in a dose-dependent manner. Both processes therefore depend on a PTK signaling cascade. Specifically, macrophage exposure to titanium-induced phosphorylation of multiple proteins including the Src kinase Lyn and phospholipase Cgamma-1 and Cgamma-2. Phosphorylation peaked within 2 min and returned to baseline within 45 min. Similar but not identical phosphorylation patterns were obtained when cells were stimulated with titanium preincubated with serum or albumin, suggesting distinct signal transduction pathways dependent on particle coating.