Osteoblastic MG-63 cell differentiation, contraction, and mRNA expression in stress-relaxed 3D collagen I gels.
To investigate the molecular aspects of osteoblastic interactions with a type I collagen matrix, human osteoblast-like MG-63 cells were cultured in three-dimensional (3D) collagen I gels. MG-63 cells in collagen gels expressed higher osteocalcin mRNA levels than cells in monolayer (2D) on polystyrene surfaces. Gel contraction was assessed via releasing the collagen gels from attachment following 24 h incubation in serum free, TGF-beta1-treated, or 1,25-(OH)(2)D(3)-treated media. 10 ng/ml of TGF-beta1 was optimal for enhancing contraction and led to decreased osteocalcin mRNA levels. In contrast, 50 nM 1,25-(OH)(2)D(3) led to increased osteocalcin mRNA levels, but did not affect contraction. Furthermore, the effect of contraction on gene expression was examined by releasing a subset of gels after 24 h and assessing mRNA levels by RT-PCR. Contracting gels exhibited temporally regulated differential increases in MMP-1, MMP-3, and alpha(2) integrin mRNA levels at specific time points post release. Cytochalasin D treatment immediately following release of gels inhibited contraction in a dose-dependent manner as well as prevented upregulation of MMP-1, MMP-3, and alpha2 integrin mRNA levels in contracting gels. These results suggest that osteoblastic cells generate internal loads that may affect specific gene expression, and these changes can be altered in the presence of biomediators.
Parreno
Justin
J
McCaig Institute for Bone and Joint Health, University of Calgary, 3330 Hospital Dr. N.W., Calgary, AB, Canada, T2N 4N1. jparreno@ucalgary.ca
Buckley-Herd
Geoff
G
de-Hemptinne
Isabelle
I
Hart
David A
DA
D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002762:Cholecalciferol / Q000494:pharmacology; D024042:Collagen Type I / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D005782:Gels; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D039421:Integrin alpha2 / Q000235:genetics / Q000378:metabolism; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D024042:Collagen Type I; D005782:Gels; D039421:Integrin alpha2; D021122:Protein Subunits; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D015675:Osteocalcin; D002762:Cholecalciferol; D015638:Cytochalasin D; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1