safranine-t and Inflammation

Topics: Animals; Benzenesulfonates; Cartilage, Articular; Inflammation; Knee Joint; Mice; Phenazines; Picrates; Staining and Labeling

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is known to inhibit matrix metalloproteinases, aggrecanases, and tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, ADAM17). These metalloproteases participate in different aspects of joint destruction in inflammatory arthritis. To determine the relative importance of this inhibitor in joint pathology, wild-type and Timp3-/- mice were immunized with methylated bovine serum albumin followed by arthritis induction by intra-articular injection of the same antigen. Animals were monitored for up to 14 days after challenge, and joint tissues were analyzed by routine and Safranin O staining and for the presence of aggrecan neoepitopes produced by metalloprotease cleavage. Serum TNF-alpha was measured by immunoassay. Compared to wild-type animals, Timp3-/- mice showed a dramatic increase in the initial inflammatory response to intra-articular antigen injection, and serum TNF-alpha levels were greatly elevated in the Timp3-/- animals after immunization. However, these differences in clinical features disappeared by days 7 to 14. No difference in Safranin O staining or aggrecan cleavage site neoepitope abundance was seen. Thus, in inflammatory joint disease TIMP-3 likely dampens the inflammatory response of TNF-alpha by reducing ADAM17 activity.

Topics: ADAM Proteins; ADAM17 Protein; Aggrecans; Animals; Antigens; Arthritis; Coloring Agents; Disease Models, Animal; Extracellular Matrix Proteins; Immunohistochemistry; Inflammation; Injections, Intra-Articular; Lectins, C-Type; Metalloendopeptidases; Mice; Mice, Knockout; Phenazines; Proteoglycans; Serum Albumin, Bovine; Tissue Inhibitor of Metalloproteinase-3; Tumor Necrosis Factor-alpha

In this study, hydrogel scaffolds, based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF), were implanted into osteochondral defects in the rabbit model. Scaffolds consisted of two layers-a bottom, bone forming layer and a top, cartilage forming layer. Three scaffold formulations were implanted to assess how material composition and transforming growth factor-beta1 (TGF-beta1) loading affected osteochondral repair. Critical histological evaluation and scoring of the quantity and quality of tissue in the chondral and subchondral regions of defects was performed at 4 and 14 weeks. At both time points, no evidence of prolonged inflammation was observed, and healthy tissue was seen to infiltrate the defect area. The quality of this tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 14 weeks. A promising degree of Safranin O staining and chondrocyte organization was observed in the newly formed surface tissue, while the underlying subchondral bone was completely integrated with the surrounding bone at 14 weeks. Material composition within the bottom, bone-forming layer did not appear to affect the rate of scaffold degradation or tissue filling. However, no bone upgrowth into the chondral region was observed with any scaffold formulation. TGF-beta1 loading in the top layer of scaffolds appeared to exert some therapeutic affect on tissue quality, but further studies are necessary for scaffold optimization. Yet, the excellent tissue filling and integration resulting from osteochondral implantation of these OPF-based scaffolds demonstrates their potential in cartilage repair strategies.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Bone and Bones; Bone Diseases; Bone Substitutes; Cartilage; Cartilage, Articular; Chondrocytes; Coloring Agents; Disease Models, Animal; Fractures, Bone; Gelatin; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Inflammation; Lipid Bilayers; Materials Testing; Osteochondritis; Phenazines; Polyesters; Polyethylene Glycols; Rabbits; Regression Analysis; Time Factors; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Within a murine model of regional immunotherapy, the cytolytic potential of peritoneal neutrophils could not be confirmed or quantified using routine techniques of cell separation and chromium release assays. We, therefore, developed procedures for the enrichment of neutrophils and estimation of the frequency of killer cells. Peritoneal exudate cells from mice injected with Corynebacterium parvum were fractionated on a self-generating Percoll gradient to enrich for neutrophils and deplete macrophages. A significant enrichment of neutrophils (greater than 90%) was obtained in a band corresponding to a density of 1.088 with a recovery of 35-50% of input. Neutrophil-enriched cell populations were then mixed with tumor cells to examine neutrophil-target interactions at the single cell level. Conjugates of neutrophils and tumor targets were obtained and the majority were lytic. With the aid of trypan blue staining and safranin counterstaining, it was possible to distinguish effector cells from targets and neutrophils from other host cells. The frequency of conjugates was dependent upon the effector to target cell ratio and was not affected by changes in temperature (range 4-30 degrees C). The post-binding lytic events were initiated rapidly after conjugation and tumor lysis was completed within 30 min. The lytic events occurred optimally between 25 degrees and 37 degrees C. The present studies support the role of neutrophils in tumor lysis following administration of an immunoadjuvant. The techniques described are important to further study the role of neutrophils in disease states as well as the underlying mechanisms of neutrophil-mediated tumor cytotoxicity.

Topics: Animals; Cell Line; Cytotoxicity Tests, Immunologic; Female; Inflammation; Killer Cells, Natural; Mice; Mice, Inbred C3H; Neoplasms, Experimental; Neutrophils; Phenazines; Povidone; Silicon Dioxide