This study demonstrated that PRP and AS both contained epitheliotrophic growth factors which are known to play important roles in cell migration, cell proliferation, and wound healing. Furthermore, this study showed that growth factors in PRP and AS were stable after storage at -20°C for at least 3 months. Moreover, we determined that PRP and AS were safe and effective in the treatment of DED.
In this study, we determined that all four epitheliotrophic growth factors of interest: EGF, fibronectin, PDGF-AB, and TGF-β1, were present in both PRP and AS eye drops as expected. However, there were some differences in concentration when compared between those in PRP and in AS (Fig. 1). Particularly, the levels of PDGF-AB was significantly higher in AS at baseline and after 2 storage conditions (Fig. 1C), while TGF- β1 was present at higher concentration in PRP at all time points (Fig. 1D). Fibronectin was present at similar level in both eye-drops at baseline but increased to be significantly higher in PRP after 2 storage conditions (Fig. 1B).
Furthermore, we ascertained that the concentrations of all four growth factors in both AS and PRP did not drop within the 3-month time frame that patients normally kept these blood-derived eye drops (Fig. 2). This result is consistent with previous studies which showed that EGF and fibronectin were stable in PRP and AS at -20°C for 3 months.24,28 However, one study reported contrasting findings, in which they found that after storage at -20°C for up to 3 months, only EGF was stable in PRGF but fibronectin was not.29 This might suggest that different preparations of blood-derived eye drop is the cause of the differences in stability and might indicate the superiority of PRP over PRGF. Our results confirm that both PRP and AS can be frozen for up to 3 months without a decrease in the levels of growth factors.
Surprisingly, when stored at 4°C, EGF, fibronectin, and PDGF-AB in PRP increased from baseline levels and fibronectin also increased in AS. Particularly, EGF in PRP increased approximately 3 times from baseline to the level which exceeded that in AS (Fig. 1A, Fig. 2A and E). Additionally, we observed that EGF in PRP increased almost 3 times from baseline after storing at -20°C as well (Fig. 2A). Increase of certain epitheliotrophic growth factors may be explained by the activation of platelets in PRP, which is composed of blood plasma enriched with platelets. Platelet activation can occur in some conditions, such as storage duration, temperature, calcium chloride addition, etc.28,30,31 Previous studies and our results suggest that it could be beneficial for patients to use PRP after it has been frozen or refrigerated rather than the fresh preparation.
Many factors contribute to the different amount of epitheliotrophic growth factors in each subject; for example, age, gender, underlying diseases, drug use, and daily activity and routine.23,32,33 The difference in PRP and AS preparation methods also contribute to the differences in growth factor levels. That may explain why our results of concentration of growth factors were different from those in some previous studies.28 When comparing with the study in human tear, we found that TGF-β1 and fibronectin levels in PRP were similar to those in human tear but EGF level in PRP was lower.13 However, our results on clinical efficacy suggest that despite some variations in growth factor concentrations, PRP is likely to have enough growth factors to be useful in treating DED patients.
This study showed the efficacy of PRP and AS in DED treatment based on improvement of symptoms and sign of DED (Table 1). PRP might be superior to AS due to its ability to significantly improve dry eye questionnaire score, OSDI score, and BCVA, whereas the improvement of only OSDI score was observed in AS-treated group when compared to baseline. For Schirmer’s test, while the improvement from treatment over baseline scores did not reach significant levels in either group, it was observed that PRP-treated eyes had significantly more improvement than AS-treated eyes when comparing between groups. These results were of similar patterns to those reported in previous studies that investigated the use of AS or PRP compared to artificial tears in DED.6,8,34 Another factor that PRP may be preferred over AS is that the preparation procedure of AS includes an additional 2-hour incubation period. In clinical practice, this additional waiting time could create inconvenience for patients and influence their preference.
The strength of this study was that it measured the levels of growth factors and their stability in the same samples prescribed to patients and analyzed for clinical efficacy. Moreover, since the clinical study design included the use of PRP and AS in each eye of the same patients, growth factor concentrations were also measured in PRP and AS of the same persons to lessen variability due to lifestyle and individual’s health. Additionally, the samples were derived from dry eye patients rather than those of healthy volunteers which might be different due to demographic and underlying diseases. However, the disadvantage of this study was that all recruited subjects were female, which may be due to the higher prevalence of DED in female.1 Therefore, the levels of epitheliotrophic growth factors in this study may be influenced by gender. The number of participants in this study was limited because we would like to test the same samples in the lab and clinical study. Future study comparing the clinical efficacy between PRP and AS may aim to recruit more patients, especially some of male gender. Additionally, it would be interesting to study which storage condition releases the most growth factors and the optimal concentration of growth factors in wound healing of ocular surface.
In conclusion, PRP could be considered as an alternative to AS for the treatment of DED because its efficacy and the level of epitheliotrophic growth factors presented were not inferior to those of AS, while PRP requiring much shorter preparation time. Moreover, this study also shows that PRP eye drops can be stored at -20ºC for up to at least 3 months without decrements of growth factors concentration.