Passage in monolayer influences the response of chondrocytes to dynamic compression.
This study tests the hypothesis that expansion by passage in monolayer influences the response of isolated articular chondrocytes to dynamic compression. Chondrocytes, isolated from bovine articular cartilage, were seeded in monolayer and passaged 4 times (P1-4). For assessment of chondrocytic and fibroblastic phenotype, freshly isolated and passaged cells were seeded on glass coverslips or in 2% alginate beads and cultured for 7 days in DMEM + 10% FCS. Samples were assayed for DNA and GAG content and stained for collagen types I and II. In separate experiments, freshly isolated or passaged chondrocytes were seeded at 10 x 10(6) cells.ml(-1) in 4% cylindrical agarose constructs and subjected to 15% dynamic compressive strain at 1 Hz for 24 hours. [(3)H]-thymidine incorporation, SO(4) incorporation and nitrite release were analysed. Immediately following isolation (P0), chondrocytes seeded in alginate expressed high levels of type II collagen, but did not stain for type I collagen. Following repeat passage the cells expressed enhanced levels of type I collagen, with an associated reduction in type II collagen staining. These data indicate a modulation to a fibroblastic phenotype during monolayer expansion which was not rapidly reversed by culture in a 3D hydrogel. Dynamic compression down-regulated SO(4) incorporation at P0, but did not affect [(3)H]-thymidine incorporation. By contrast the incorporation of both SO(4) and [(3)H]-thymidine was enhanced by dynamic compression at both P1 and to a lesser extent P2. SO(4) and [(3)H]-thymidine incorporation were inhibited at P3 and P4. Nitrite release was down-regulated by dynamic compression at all passages. These data demonstrate a clear modulation in the response of bovine articular chondrocytes to dynamic compression following passage in monolayer.
Wiseman
Mike
M
Institute of Orthopaedics, University College London Medical School, Brockley Hill, Stanmore, Middlesex HA7 4LP, UK.
Bader
Dan L
DL
Reisler
Tom
T
Lee
David A
DA
D002358:Cartilage, Articular / Q000502:physiology*; D018929:Cell Culture Techniques; D002469:Cell Separation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D024042:Collagen Type I / Q000032:analysis; D024043:Collagen Type II / Q000032:analysis; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D011312:Pressure; D013431:Sulfates / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D024042:Collagen Type I; D024043:Collagen Type II; D013431:Sulfates; D013936:Thymidine