Effect of two different preparations of platelet-rich plasma on synoviocytes

Abstract

Purpose: To analyse the modifications induced by two different platelet-rich plasma (PRP) preparations on osteoarthritis (OA) synoviocytes, by documenting changes in gene expression of factors involved in joint physiopathology.

Methods: OA synoviocytes were cultured for 7 days in medium with different concentrations of either P-PRP (a pure platelet concentrate without leucocytes but with a limited number of platelets), L-PRP (a higher platelet concentrate containing leucocytes) or platelet-poor plasma (PPP). Gene expression of interleukin (IL)-1beta, IL-6, IL-8/CXCL8, tumour necrosis factor alpha, IL-10, IL-4, IL-13, metalloproteinase-13, tissue inhibitor of metalloproteinase (TIMP)-1, (TIMP)-3, (TIMP)-4, vascular endothelial growth factor, transforming growth factor beta1, fibroblast growth factor (FGF)-2, hepatocyte growth factor (HGF), hyaluronic acid (HA) synthases (HAS)-1, (HAS)-2, and (HAS)-3 was analysed by RT-PCR. HA production was determined in culture supernatants by ELISA.

Results: IL-1β, IL-8 and FGF-2 were significantly induced by L-PRP compared to both P-PRP and PPP; HGF was down-modulated by L-PRP versus both P-PRP and PPP, and an inverse dose-response influence was shown for all preparations. Expression level of TIMP-4 was lower in the presence of L-PRP compared with P-PRP. HA production and HAS gene expression did not seem to be modulated by PRP.

Conclusions: L-PRP is able to sustain the up-regulation of proinflammatory factors, (IL-1beta, IL-8 and FGF-2), together with a down-modulation of HGF and TIMP-4 expression, two factors that have been recognized as anti-catabolic mediators in cartilage, thus supporting the need to further optimize the PRP preparations to be applied in clinical practice.

Fig. 1

Gene expression analysis of interleukin (IL)-1beta, IL-8/CXCL8, IL-6, IL-10, tumour necrosis factor (TNF) alpha. Synovial fibroblasts were treated for 7 days with 5, 10, 20 % of L-PRP, P-PRP or PPP obtained from each subject (n = 7). Gene expression relative quantification was performed, and data are expressed as number of molecules *100,000 GAPDH. Boxes indicate the 25 and 75 % percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median; p value significances are shown in tables beside each figure, as determined by General Linear Model statistical analysis and Kendall Tau correlation; ns not significant

Fig. 2

Gene expression analysis of hepatocyte growth factor (HGF), fibroblast growth factor (FGF)-2, transforming growth factor (TGF) beta, vascular endothelial growth factor (VEGF). Synovial fibroblasts were treated for 7 days with 5, 10, 20 % of L-PRP, P-PRP or PPP obtained from each subject (n = 7). Gene expression relative quantification was performed, and data are expressed as number of molecules *100,000 GAPDH. Boxes indicate the 25 and 75 % percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median; p value significances are shown in tables beside each figure, as determined by General Linear Model statistical analysis and Kendall Tau correlation; ns not significant

Fig. 3

Gene expression analysis of cartilage matrix-degrading enzyme metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase inhibitors (TIMP-1,-3,-4). Synovial fibroblasts were treated for 7 days with 5, 10, 20 % of L-PRP, P-PRP or PPP obtained from each subject (n = 7). Gene expression relative quantification was performed, and data are expressed as number of molecules *100,000 GAPDH. Boxes indicate the 25 and 75 % percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median; p value significances are shown in tables beside each figure, as determined by General Linear Model statistical analysis; ns not significant

Fig. 4

Hyaluronic acid modulation by PRP. Synovial fibroblasts were treated for 7 days with 5, 10, 20 % of L-PRP, P-PRP or PPP obtained from each subject (n = 7). Hyaluronic acid synthases 1-2-3 gene expression relative quantification was performed, and data are expressed as number of molecules *100,000 GAPDH. Hyaluronic acid protein production was normalized per number of cells. Boxes indicate the 25 and 75 % percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median; p value significances are shown in tables beside each figure, as determined by General Linear Model statistical analysis and Kendall Tau correlation; ns not significant