249 Lyophilised platelet-rich plasma stimulates migration of equine endometrial mesenchymal stem and epithelial cells

Reproductive diseases have a strong impact on the economics of the horse industry. Endometriosis is a major cause of equine infertility; it involves poor tissue renovation, impaired cell migration, and decreased vascularisation. Endometritis is an inflammatory disease after artificial insemination or post-breeding. There is no efficient treatment for the former; for the latter, the existing treatments are based on antibiotics to reduce inflammation and contamination of the uterus, but they are not effective. Platelet-rich plasma (PRP) is a blood derivative containing growth factors that can act in injured tissues by mitogenic, neovascular, and anti-inflammatory effects. We have used PRP for the treatment of endometritis with encouraging results. Mares with endometritis receiving local PRP showed a downregulation of intrauterine inflammation. Autologous PRP for endometritis has been used, but it is neither practical nor economically feasible; thus, other sources of PRP are welcome. Lyophilisation of PRP (FD-PRP) is an alternative for this problem. We hypothesise that FD-PRP maintains its biological attributes and, in vitro, improves epithelial and mesenchymal stem cell (MSC) migration compared to fresh PRP. PRP was produced locally, according to our standard procedure. The enrichment of platelet preparations was made by counting them before and after the PRP preparation. FD-PRP was made in a freeze-dryer machine (Lyovapor™ L-200, BUCHI) and stored at 4°C for one month. Horse heterologous equine mesenchymal stem cells (eMSC) and epithelial cells were isolated from endometrium, characterised in the laboratory, and used in passages 3 to 5. Migration was evaluated by the scratch assay, for which cells were seeded in p12 wells, and were exposed at at 90% confluency to mitomycin C (10 µg/mL) for 2 h to avoid proliferation. Scratch was made with a 200-µL tip; photographs were taken at time 0 and 36 h after. Percentage of filling the scratched area was the criterion of success and it was calculated by ImageJ Software (National Instititues of Health). Experimental groups included DMEM medium +5% v:v of reconstituted FD-PRP or fresh PRP. Positive control: same medium +10% fetal calf serum (FCS). Negative control: DMEM + 1% FCS. All experiments were performed using three technical and four biological replicates. ANOVA test and post hoc Tukey were performed with P < 0.05. PRP and FD-PRP induced faster migration of eMSC versus positive control: eMSC positive control 50 ± 6% filling of the scratch at 36 h, negative control: 30 ± 7%, PRP: 75 ± 7%; FD-PRP 86 ± 8%. For epithelial cells: 100, 64 ± 5%; 88 ± 2% and 95 ± 2% (for positive, negative controls, PRP, and FD-PRP respectively). Our results indicate that in vitro PRPs enhance the migration of eMSC and epithelial cells, due to growth factors released by platelets. To our knowledge, this is the first report of FD-PRP in vitro in cells of equine endometrium. This opens the opportunities for testing the FD-PRP as novel therapeutic tool in reproductive problems in mares. FD-PRP preserves the biological activity of PRP while providing a stock of autologous or heterologous biologicals for the treatment of endometrial diseases at any time.

This research was supported by Fondecyt Regular 1210349.