Proliferating and differentiating effects of three different growth factors on pluripotent mesenchymal cells and osteoblast like cells

Abstract

Growth factors are in clinical use to stimulate bone growth and regeneration. BMP-2 is used in long bone and spinal surgery, PDGFbb for the treatment of periodontal defects and children with growth hormone receptor deficiency are treated with IGF-I.Aim of the present study was the comparative analysis of the effect of these growth factors released from a local drug delivery system on cells of the osteogenic lineage at differing differentiation stages.The experiments with the mesenchymal cell line C2C12 revealed a proliferating effect of all three growth factors and a differentiating effect of BMP-2 with a dramatic increase in alkaline phosphatase activity. None of the growth factors stimulated cell migration.Human osteoblast like cells showed similar results with an increase in proliferation after stimulation with IGF-I or PDGFbb. The enzymatic activity of alkaline phosphatase was enhanced only in the cells stimulated with BMP-2. This group showed also more mineralized matrix compared to the other groups.In conclusion, the growth factors IGF-I and PDGFbb delivered with a local drug delivery system stimulated cell proliferation, whereas BMP-2 showed a dramatic effect on differentiation on osteoblast precursor cells and osteoblast like cells.

Figure 1

a) Cell count of the osteoblast like cell culture treated with different growth factors. The data presented are normalized to the control group (PDLLA) which is set 100%. A significant increase in the cell number was seen after treatment with PDGFbb or IGF-I (days 2–10) in comparison to the PDLLA treated cells (ANOVA, Dunnett). b) Alkaline phosphatase activity (AP) of the osteoblast like cell culture treated with different growth factors. The data presented are normalized to the control group (PDLLA) which is set 100%. A significant increase in AP activity was seen after treatment with BMP-2 (days 5–15) in comparison to the PDLLA treated cells (ANOVA, Dunnett).

Figure 2

a-d) hOB 15 days after culturing with different growth factors stained with a combination of AP (blue) and v. Kossa. An intense mineralization is detectable in the osteoblast like cells treated with BMP-2 (d). e-h) C2C12 cells stained for alkaline phosphatase. The pluripotent mesenchymal cell line treated with BMP-2 (h) showed an intense blue alkaline phosphatase staining.

Figure 3

a) Cell count of the pluripotent mesenchymal cell line (C2C12) treated with different growth factors. The data presented are normalized to the control group (PDLLA) which is set 100%. A significant increase in the cell number was seen after treatment with PDGFbb, IGF-I or BMP-2 in comparison to the PDLLA treated cells (ANOVA, Dunnett). b) Alkaline phosphatase activity (AP) of pluripotent mesenchymal cell line (C2C12) treated with different growth factors. The data presented are normalized to the control group (PDLLA) which is set 100%. A significant increase in AP activity was seen after treatment with BMP-2 in comparison to the PDLLA treated cells (ANOVA, Dunnett). c) Migration assay of C2C12 cells. A significant migration was detectable in the control group (10% FCS) but not in the growth factor groups.