ABSTRACT
Dedifferentiated human articular chondrocytes exhibited a wide variation in their capacity to proliferate and redifferentiate in an alginate suspension culture system. The greatest extent of proliferation and redifferentiation was seen to be dependent on the formation of clonal populations of chondrocytes and correlated inversely with the initial cell seeding density. Redifferentiating chondrocytes seeded at low density (1 x 10(4) cells/ml alginate) compared with chondrocytes that were seeded at high density (1 x 10(6) cells/ml alginate) showed a nearly 3-fold higher median increase in cell number. a 19-fold greater level of type-II collagen mRNA expression, a 4-fold greater level of aggrecan mRNA expression, and a 6-fold greater level of sulfated glycosaminoglycan deposition at 4 weeks of culture. Matrix molecules from low-density cultures were assembled into chondrocyte-encapsulated, spherical extracellular matrices that were readily visualized in sections from 12-week cultures stained with antibodies against types I and II collagen and aggrecan. Ultrastructural analysis of 12-week low-density cultures confirmed the presence of thin collagen fibrils throughout the matrix.
Subject(s)
Alginates/pharmacology , Cartilage, Articular/drug effects , Cell Differentiation/drug effects , Cell Division/drug effects , Chondrocytes/drug effects , Culture Media/pharmacology , Extracellular Matrix Proteins , Adult , Aggrecans , Cartilage, Articular/growth & development , Cartilage, Articular/ultrastructure , Cell Culture Techniques , Cell Differentiation/physiology , Cell Division/physiology , Chondrocytes/metabolism , Chondrocytes/ultrastructure , Collagen/genetics , Collagen/metabolism , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Female , Gene Expression Regulation, Developmental/physiology , Glycosaminoglycans/metabolism , Humans , Lectins, C-Type , Male , Middle Aged , Proteoglycans/genetics , Proteoglycans/metabolism , RNA, Messenger/metabolism , Sulfates/metabolismABSTRACT
A technique for measurement of retrograde coronary blood flow in intact anesthetized dogs is described. Occlusion of the coronary artery is produced by the inflation of a small rubber balloon at the tip of a no. 9 cardiac catheter placed under fluorescopy in a branch of the left coronary artery. Blood which bleeds back from the occluded coronary artery through the no. 9 catheter is diverted into a small reservoir of 1-ml capacity. The time to fill this reservoir is recorded electrically. Retrograde coronary blood flow is calculated from the time required to fill this reservoir. Results indicate good repeatability of meadurements. The technique seems to be a simple, adequate, and convenient means for assessing agents for possible vasodilator action on the collateral circulation in intact animals.