In Vitro Effects of PDGF Isoforms (AA, BB, AB and CC) on Migration and Proliferation of SaOS-2 Osteoblasts and on Migration of Human Osteoblasts

Abstract

PDGF is a major constituent of platelet rich plasma (PRP), responsible of chemotactic and possibly of mitogenic effects of PRP on osteoblasts. PDGF family includes 5 isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, all expressed in platelets except PDGF-DD. Aim of this study was to analyze the effect of recombinant hPDGF-A, -AB, -B and -C, on migration and proliferation of a human osteoblastic cell line, SaOS-2. Preliminary observations on cell migration were also done in primary cultures of human osteoblasts. In vitro microchemotaxis and (3)H-thymidine mitogenic assays were used. While PDGF-AB is active at concentrations present in PRP, PDGF-AA and BB are chemotactic only at much higher doses. PDGF-C is totally inactive alone or together with the active isoforms. PDGF-AA, PDGF-BB and PDGF-C stimulate SaOS-2 proliferation only at the highest dose tested, while PDGF-AB is ineffective. Primary osteoblasts are less sensitive than SaOS-2 and progressively lose responsiveness with increasing passages in culture, in line with loss of cell differentiation. The different PDGF isoforms act differentially on osteoblasts, the-AB isoform appearing the major responsible of the PRP chemiotaxis. PDGF, at the concentrations present in PRP, does not affect cell proliferation.

Keywords: PDGF isoforms;; PRP; SaOS-2-cells; migration; osteoblasts.

Figure 1

Expression of PDGF receptor alpha and beta in SaOS-2 cells. Data are the Ct values of each standard point. The line represents the regression curve.

Figure 2

Effect of different PDGF isoforms on SaOS-2 cells migration. Values are means ± SD of the rate of migrated cells compared to DMEM. The number of samples analyzed is shown in the squares at the bottom of the columns. *p<0.05 vs. DMEM.

Figure 3

Effect of different PDGF-C doses on SaOS-2 cells migration. Values are means ± SD of the rate of migrated cells compared to DMEM. The number of samples analyzed is shown in the squares at the bottom of the columns.

Figure 4

Effect of different PDGF isoforms on SaOS-2 cells proliferation. Values are means ± SD of the rate of [³H]-thymidine incorporation compared to DMEM. The number of samples analyzed is shown in the squares at the bottom of the columns. *p<0.05 vs. DMEM.

Figure 5

Effect of different PDGF-C doses on SaOS-2 cells proliferation. Values are means ± SD of the rate of [³H]-thymidine incorporation compared to DMEM. The number of samples analyzed is shown in the squares at the bottom of the columns. *p<0.05 vs. DMEM.

Figure 6

Photomicrographies of primary cultures of human osteoblasts, labelled with osteopontin (OPN). Panels a and b: from 2nd to 4th passage in culture the presence of OPN, a phenotypic marker for osteoblasts, is indicative of a good differentiation of these cells as osteoblasts. Panels c and d: from the 5th passage in vitro, the OPN positive cells show a progressive loss of differentiation. Panels a and d: original magnification 40×; panels b and c: original magnification 20×.

Figure 7

Sensitivity of primary human osteoblasts to the chemotactic effect of PRP after different passages in culture. Values are means ± SD of the rate of migrated cells compared to DMEM.

Figure 8

Effect of different PDGF isoforms on primary human osteoblast migration. Values are means ± SD of the rate of migrated cells compared to DMEM. The number of samples analyzed is shown in the squares at the bottom of the columns. *p<0.05 vs. DMEM