An elevated number of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a gradient isolation procedure.
Bone marrow stromal cells (BMSCs) for osteoblast differentiation studies can be obtained by gradient isolation techniques or by directly plating a filtered cell suspension. We compared these two procedures to evaluate whether this step is critical in order to obtain a high number of differentiated colonies. Isolated primary rat BMSCs were cultured in vitro with or without insulin-like growth factor II (IGFII), basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF) or transforming growth factor beta 1 (TGF beta 1), and histochemically and biochemically analysed at different time points. The gradient procedure produced a significantly higher number of colonies capable of osteoblastic differentiation. The growth factors had different effects. In particular, b-FGF and EGF significantly increased the number of Alizarin red S positive colonics, while IGFII and TGF beta I exerted inhibitory effects. Nodules obtained on day 21 showed some alkaline phosphatase positive cells and were Von Kossa-positive. These data demonstrate that more differentiated colonies are obtainable from BMSCs isolated by the gradient procedure.
Lisignoli
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Laboratorio di Immunologia e Genetica, I.O.R., Via di Barbiano 1/10, 40136 Bologna, Italy.
Remiddi
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G
Cattini
L
L
Cocchini
B
B
Zini
N
N
Fini
M
M
Grassi
F
F
Piacentini
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A
Facchini
A
A
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects; D002452:Cell Count; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D008297:Male; D010006:Osteoblasts / Q000166:cytology*; D051381:Rats; D011916:Rats, Inbred F344; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor; D007335:Insulin-Like Growth Factor II; D000469:Alkaline Phosphatase