Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1.
Cell culture procedures were developed for use with surgical and normal control specimens of the annulus of the human intervertebral disc. Cells were established in monolayer explant culture and seeded into three-dimensional growth environments of alginate or agarose; under these growth conditions cells assumed a rounded phenotype and formed colonies. A novel method of layering suspensions of cells onto cell well inserts proved technically much easier than the microbead culture method. Immunohistochemistry was utilized to demonstrate in vitro production of the following extracellular matrix components: types I, II, III, and VI collagen, 4-S-chondroitin sulfate, and keratan sulfate. Young and old age- and gender-matched cells grown in the presence of TGF-beta1 showed significant enhancement of proliferation after 4 days of exposure to TGF-beta with a lessened mitogenic response present after 10 days. Molecular studies of proteoglycan gene expression showed that at 4 days young normal cells had increased biglycan, but not decorin, message levels. Decorin expression was unchanged at Day 4 and decreased or shut off by Day 10. Results support the use of three-dimensional culture systems for in vitro evaluation of human disc cell function and expand our understanding of the in vitro behavior of these cells.
Gruber
H E
HE
Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, North Carolina 28232, USA. hgruber@carolinas.org
Fisher
E C
EC
Desai
B
B
Stasky
A A
AA
Hoelscher
G
G
Hanley
E N
EN
D000328:Adult; D000464:Alginates; D058578:Biglycan; D001672:Biocompatible Materials; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000032:analysis / Q000096:biosynthesis; D003094:Collagen / Q000032:analysis / Q000096:biosynthesis*; D058575:Decorin; D016326:Extracellular Matrix Proteins; D005260:Female; D015870:Gene Expression; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006801:Humans; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D007632:Keratan Sulfate / Q000032:analysis / Q000096:biosynthesis; D007700:Kinetics; D011509:Proteoglycans / Q000096:biosynthesis*; D012685:Sepharose; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000464:Alginates; C546884:BGN protein, human; D058578:Biglycan; D001672:Biocompatible Materials; C546878:DCN protein, human; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006603:Hexuronic Acids; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; D020723:Glucuronic Acid; D002809:Chondroitin Sulfates; D003094:Collagen; D012685:Sepharose; D007632:Keratan Sulfate