Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production

Abstract

Purpose: Platelet-rich plasma (PRP) contains growth factors and creates a 3D structure upon clotting; PRP or platelet lysate (PL) might be considered for annulus fibrosus (AF) repair.

Methods: Bovine AF cells were cultured with 25% PRP, 50% PRP, 25% PL, 50% PL, or 10% FBS. After 2 and 4 days, DNA, glycosaminoglycan (GAG), and mRNA levels were analyzed. Histology was performed after injection of PRP into an AF defect in a whole disc ex vivo.

Results: By day 4, significant increases in DNA content were observed in all treatment groups. All groups also showed elevated GAG synthesis, with highest amounts at 50% PL. Collagen I and II expression was similar between groups; aggrecan, decorin, and versican expression was highest at 25% PL. Injection of PRP into the AF defect resulted in an increased matrix synthesis.

Conclusions: Platelet-rich preparations increased the matrix production and cell number and may therefore be considered to promote AF repair.

Figures

Fig. 1

Representative light microscopy images of AF cells in different media at day 2; scale bar 200 μm. (a), (b) AF cells treated with PRP. The images were taken at the same location at different focuses, indicating that some of the cells were attached to the bottom of the culture dish, while others were embedded in the viscous medium. Within this gel-like structure, AF cells adopted a more rounded phenotype and were arranged in colonies. (c) AF cells in PL containing medium. Whereas the cells appeared spindle-shaped like in the other monolayer culture conditions, they did form cell conglomerations. (d) AF cells in FBS containing medium. The cell conformation in PPP, FBS, and DMEM media was similar

Fig. 2

(a) DNA and (b) GAG content of whole samples after 2 and 4 days of cell culture with platelet-rich plasma (25 and 50 % PRP), platelet-lysate preparation (25 and 50 % PL), platelet-poor plasma (50 % PPP), fetal bovine serum (10 % FBS), and DMEM (100 % DMEM). Asterisk indicates a significant difference (P < 0.05) when compared with 100 % DMEM. Mean + SEM, n = 5, triplicates per group

Fig. 3

Relative mRNA expression of AF cells after 2 and 4 days of culture with platelet-rich plasma (25 and 50 % PRP), platelet-lysate preparation (25 and 50 % PL) and platelet-poor plasma (50 % PPP). (a) collagen type I (COL1), (b) collagen type II (COL2), (c) aggrecan (ACAN), (d) versican (VCAN), (e) biglycan (BGN), and (f) decorin (DCN) are shown. Data were normalized to the values of freshly isolated cells (day 0). Asterisk indicates a significant difference (P < 0.05) as compared to the 50 % PPP group. Mean + SEM, n = 4, triplicates per group

Fig. 4

Representative images of the whole disc model after Safranin-O staining; scale bar 200 μm. (a) Cut through the AF without addition of PRP (control). (b) Injured AF and injection of PRP. (c) The area adjacent to the cut; sample without PRP. (d) The area adjacent to the cut, after PRP injection