Three-dimensional culture of human meniscal cells: extracellular matrix and proteoglycan production.
BACKGROUND
The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-beta (TGF-beta).


METHODS
Experimental studies were approved prospectively by the authors' Human Subjects Institutional Review Board. Human meniscal cells were isolated from surgical specimens, established in monolayer culture, seeded into a 3D scaffold, and cell morphology and extracellular matrix components (ECM) evaluated either under control condition or with addition of TGF-beta. Outcome variables were evaluation of cultured cell morphology, quantitative measurement of total sulfated proteoglycan production, and immunohistochemical study of the ECM components chondroitin sulfate, keratan sulfate, and types I and II collagen.


CONCLUSIONS
Meniscal cells attached well within the 3D microenvironment and expanded with culture time. The 3D microenvironment was permissive for production of chondroitin sulfate, types I and II collagen, and to a lesser degree keratan sulfate. This microenvironment was also permissive for growth factor responsiveness, as indicated by a significant increase in proteoglycan production when cells were exposed to TGF-beta (2.48 microg/ml +/- 1.00, mean +/- S.D., vs control levels of 1.58 +/- 0.79, p < 0.0001). Knowledge of how culture microenvironments influence meniscal cell ECM production is important; the collagen sponge culture methodology provides a useful in vitro tool for study of meniscal cell biology.
Gruber
Helen E
HE
Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC, USA. helen.gruber@carolinashealthcare.org
Mauerhan
David
D
Chow
Yin
Y
Ingram
Jane A
JA
Norton
H James
HJ
Hanley
Edward N
EN
Sun
Yubo
Y
D018929:Cell Culture Techniques / Q000295:instrumentation; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D005781:Gelatin Sponge, Absorbable; D006801:Humans; D007632:Keratan Sulfate / Q000378:metabolism; D008592:Menisci, Tibial / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D011509:Proteoglycans / Q000187:drug effects / Q000378:metabolism*; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods*; D054457:Tissue Scaffolds; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D024042:Collagen Type I; D024043:Collagen Type II; D011509:Proteoglycans; D016212:Transforming Growth Factor beta; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate