Persistence of collagen type II synthesis and secretion in rapidly proliferating human articular chondrocytes in vitro.
Articular chondrocytes (AC) expanded in vitro for tissue engineering rapidly turn off collagen type II (COL2) synthesis. We wanted to inhibit this process sufficiently to obtain therapeutically useful numbers of AC without losing COL2 synthesis. To this end, AC were expanded on their own extracellular matrix (ECM) in structures designated chondrocytes in autologous ECM (CA-ECM). Here, AC maintained a rounded shape and proliferated rapidly. After 13-15 days in culture, 40 x 10(6) cells (median) could be obtained from a cartilage biopsy. Real-time RT-PCR showed a reduced, but persistent, production of COL2A1 mRNA at this time. Flow cytometry showed high levels of intracellular COL2, and immunogold electron microscopy showed high density of well-organized COL2 fibrils in newly synthesized ECM. Interestingly, high levels of COL1A1 mRNA and intracellular protein were detected, but no COL1 was found in the ECM. The slow loss of COL2A1 mRNA was paralleled by a loss of the COL2 regulating transcription factor SOX9 mRNA. Chromatin immunoprecipitation assays could not identify epigenetic histone modifications that would explain the observed changes in COL2 synthesis. Thus, the CA-ECM strategy allows AC to proliferate to clinically useful numbers while maintaining COL2 synthesis and secretion. This strategy may improve tissue engineering of joint surfaces.
Shahdadfar
Aboulghassem
A
Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway.
Løken
Sverre
S
Dahl
John Arne
JA
Tunheim
Siv H
SH
Collas
Philippe
P
Reinholt
Finn P
FP
Engebretsen
Lars
L
Brinchmann
Jan E
JE
D000328:Adult; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008875:Middle Aged; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics
C499177:COL2A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006657:Histones; D012333:RNA, Messenger