ABSTRACT
PURPOSE: To gain knowledge of the repair tissue in critically sized cartilage defects using bone marrow stimulation combined with CARGEL Bioscaffold (CB) compared with bone marrow stimulation (BMS) alone in a validated animal model. METHODS: Six adult Göttingen minipigs received two chondral defects in each knee. The knees were randomized to either BMS combined with CB or BMS alone. The animals were euthanized after 6 months. Follow-up consisted of histomorphometry, immunohistochemistry, semiquantitative scoring of the repair tissue (ICRS II), and µCT of the trabecular bone beneath the defect. RESULTS: There was significantly more fibrocartilage (80% vs 64%, p = 0.04) and a trend towards less fibrous tissue (15% vs 30%, p = 0.05) in the defects treated with CB. Hyaline cartilage was only seen in one defect treated with CB and none treated with BMS alone. For histological semiquantitative score (ICRS II), defects treated with CB scored lower on subchondral bone (69 vs. 44, p = 0.04). No significant differences were seen on the other parameters of the ICRS II. Immunohistochemistry revealed a trend towards more positive staining for collagen type II in the CB group (p = 0.08). µCT demonstrated thicker trabeculae (p = 0.029) and a higher bone material density (p = 0.028) in defects treated with CB. CONCLUSION: Treatment of cartilage injuries with CARGEL Bioscaffold seems to lead to an improved repair tissue and a more pronounced subchondral bone response compared with bone marrow stimulation alone. However, the CARGEL Bioscaffold treatment did not lead to formation of hyaline cartilage.
ABSTRACT
We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16. Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions. These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu. Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced. The fusion proteins were purified and analyzed as thrombin inhibitors and substrates. All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen. We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins. The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release. The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates. The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50. These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding.
Subject(s)
Fibrinogen/metabolism , Fibrinopeptide A/metabolism , Recombinant Fusion Proteins/metabolism , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Amino Acids/analysis , Base Sequence , Catalysis , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Fibrinopeptide A/genetics , Kinetics , Molecular Sequence Data , Mutation , Plasmids , Recombinant Fusion Proteins/genetics , Substrate SpecificityABSTRACT
The rate of hemolysis and the decline in glutathione (GSH) in rabbit erythrocytes caused by copper (Cu) ions were determined. Prior investigations have proposed that the oxidative stress induced by Cu ion depleted the normal cell protective mechanisms. The decline in GSH has been proposed as a necessary prerequisite for hemolysis. We have observed that both GSH decline and hemolysis are Cu dependent, but are two concurrent and independent processes. We have confirmed that oxygen is a necessary reactant for hemolysis and responsible for a major portion of GSH decline. However, in the presence of Cu ion, a slow decline in GSH occurs even in a deaerated system.