A neocartilage ideal for extracellular matrix macromolecule immunolocalization.
A neocartilage construct readily amenable to microscopy and biomechanical studies is described. Porcine articular cartilage was digested with a mixture of dispase and collagenase for chondrons or pronase and collagenase for chondrocytes. Chondrons or chondrocytes plated in 96-well plates were fixed and immunolabeled in situ for fluorescence microscopy at days 4 and 11. Collagen types I and II, aggrecan, and MMP-13 expression was assayed by semiquantitative RT-PCR. Cell numbers were analyzed by MTT assay. Chondrons and chondrocytes produced neocartilage that could be handled with minimal tearing on day 3 and none on day 11. Some cell division occurred between days 4 and 7. In both cultures, chondrocytes were surrounded by a thin rim of type VI collagen and osteopontin. Type II collagen, keratan sulfate, and tenascin were abundant throughout. At day 3, cells were rounded but by day 11 flattened cells were visible in the substratum. Continued synthesis of aggrecan and type II collagen mRNA indicated maintenance of the chondrocyte phenotype. The neocartilage was easy to immunolabel in situ without the need for sectioning, and individual cells were readily observed by microscopy. The versatility of these constructs makes them ideal for microscopy and for biomechanical studies.
Thurston Arthritis Research Center and Department of Orthopedics, University of North Carolina at Chapel Hill, NC 27599, Chapel Hill, USA.
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Parikh
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Lee
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Tchivilev
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Graff
D053674:Aggrecans; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D024042:Collagen Type I / Q000032:analysis / Q000235:genetics; D024043:Collagen Type II / Q000032:analysis / Q000235:genetics; D017364:Collagenases / Q000032:analysis / Q000235:genetics; D004396:Coloring Agents; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D037181:Lectins, C-Type; D053509:Matrix Metalloproteinase 13; D011509:Proteoglycans / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D013778:Tetrazolium Salts; D013844:Thiazoles
D053674:Aggrecans; D024042:Collagen Type I; D024043:Collagen Type II; D004396:Coloring Agents; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D013778:Tetrazolium Salts; D013844:Thiazoles; D017364:Collagenases; D053509:Matrix Metalloproteinase 13; C022616:thiazolyl blue