Fibroblastic response to treatment with different preparations rich in growth factors

Abstract

Objectives: Preparations rich in growth factors (PRGF) release them plus bioactive proteins at localized sites, with the aim of triggering healing and regenerative processes. The prevailing paradigm suggests that their influence on proliferation, angiogenesis and the extracellular matrix synthesis is minimal. However, variations in their composition and impact on different cell phenotypes have not been examined.

Materials and methods: Sixteen fibroblast cultures obtained from three different anatomical sites (skin, synovium and tendon) of 16 donors were exposed to the molecular pool released from PRGF scaffolds, with increasing amounts of platelets. We evaluated cell proliferation, secretion of angiogenic growth factors (VEGF and HGF), synthesis of type I collagen and hyaluronic acid (HA), considering platelet dose and anatomical origin of the cells. Activity of transforming growth factor-beta (TGF-beta) in type I procollagen and HA synthesis was examined by adding exogenous TGF-beta to plasma preparations.

Results: All plasma preparations induced a significant proliferative response compared to non-stimulated cells (P < 0.05). Maximum proliferation rate was obtained with PRGF with 2-fold or 4-fold platelet concentration. Exposure to PRGF stimulated VEGF synthesis exclusively in tendon cells (P < 0.05), which also exhibited a different pattern of HGF production (P < 0.05). PRGF enhanced HA synthesis (P < 0.05), but did not alter collagen I production. Platelet-secreted TGF-beta may be involved in HA, but not in type I procollagen synthesis.

Conclusions: Optimizing composition and use of platelet-rich products is crucial to enhancing the therapeutic potential of this technology. Our data show that the biological effects of PRGF may depend on concentration of platelets and on the anatomical source of the cells.

Figure 1

Fibroblasts isolated from diverse anatomical sites (skin, synovium and tendon). Representative phase contrast photomicrographs show the typical shape of diverse fibroblasts cultured on a plastic surface, and immunofluorescence microscopy confirmed that the fibroblastics were uniformly positive for prolyl 4‐hydroxylase and CD90, as revealed by immunostaining. Blue, Hoechst; green, prolyl 4‐hydroxylase and CD90.

Figure 2

Effect of plasma preparations (platelet poor, PRGF2x and PRGF4x) on proliferation of fibroblasts from the skin, synovium and tendon. Cells were seeded at a density of 20 000 cells/cm², and treated for 72 h with 20% of the supernatants released from platelet‐poor (PPP, light grey) and preparation rich in growth factors (PRGF_2x, dark grey; PRGF_4x, hatched bars) matrices. Box plot representation based on the median (line across the box) and 25th and 75th percentiles. Data summarize combined values obtained for different cell donors (skin, n = 6; synovium, n = 4; and tendon, n = 6). *P < 0.05 comparing with non‐stimulated cells; ^# P < 0.05 comparing with platelet‐poor preparation; §P < 0.05 comparing with PRGF_2x.

Figure 3

Effect of plasma preparations (platelet poor, PRGF_2x and PRGF_4x) on the secretion of angiogenic factors (VEGF and HGF) from skin, synovial and tendon fibroblast. Box plot representation based on the median (line across the box) and 25th and 75th percentiles. Grey boxes represent the group of experiments performed with the different plasma preparations: platelet‐poor (PPP, light grey) and preparation rich in growth factors (PRGF_2x, dark grey; or PRGF_4x, hatched bars). ^* P < 0.05 compared to non‐stimulated cells (NS); ^# P < 0.05 compared to platelet‐poor preparation; ^§ P < 0.05 compared to PRGF_2x.

Figure 4

Effect of plasma preparations (platelet poor, PRGF_2x and PRGF_4x) on secretion of hyaluronic acid (HA) and type I procollagen by skin, synovial and tendon fibroblasts. Box plot representation based on the median (line across the box) and 25th and 75th percentiles. Grey boxes represent the group of experiments performed with the different plasma preparations: platelet‐poor (PPP, light grey) and preparation rich in growth factors (PRGF_2x, dark grey; or PRGF_4x, hatched bars) *P < 0.05 compared to non‐stimulated cells (NS); #P < 0.05 compared to platoelet‐poor preparation; ^§ P < 0.05 compared to PRGF_2x.