Beneficial Effects of Plasma Rich in Growth Factors (PRGF) Versus Autologous Serum and Topical Insulin in Ocular Surface Cells

Abstract Purpose In the last few decades, several blood derived products such as platelet-rich plasma (PRP), plasma rich in growth factors (PRGF) and autologous serum (AS) have been used for the treatment of ocular surface disorders. Recently, insulin has been proposed to be used as an alternative for the treatment of ocular surface diseases. The aim of this study was to evaluate the biological potential of PRGF eye drops in comparison with AS and insulin on ocular surface cells. Methods Blood from three healthy young donors was collected to obtain autologous serum (AS) eye drops and plasma rich in growth factors (PRGF) eye drops. Insulin (Actrapid®) was diluted at 1 and 0.2 IU/mL. The biological potential of PRGF, AS and insulin was assessed by proliferation in HCE, HK and HConF cells. Wound healing assay was performed in HCE cells after incubation with the different treatments. HConF and HK cells were differentiated to myofibroblast after treatment with 2.5 ng/mL of TGF-β1 and then incubated with all treatments. Results PRGF eye drops induced significantly higher proliferation rate compared to AS or insulin in HConF and HK cells, but not in HCE cells. In addition, the percentage of wound healing area was significantly reduced after PRGF treatment in comparison with AS or insulin treatment. Furthermore, PRGF significantly reduced the number of myodifferentiated cells compared to AS and insulin at both concentrations analyzed. Conclusion The results obtained in the present study show that PRGF increases the biological activity of the ocular surface cells and reduces the expression of fibrosis marker compared to insulin or AS. Translational relevance The present study suggests that plasma rich in growth factors eye drops are a more effective therapy than insulin and autologous serum eye drops for the treatment of ocular surface diseases.


Introduction
Corneal disorders include a wide variety of pathologies, such as persistent epithelial defects, dry eye, limbal stem cell deficiency and ulcers, among others. A mild condition of any of these diseases may only affect the corneal and conjunctival epithelia, however progression of the eye disorder to a more severe condition may reach the corneal or conjunctival stroma, affecting the subpopulation of keratocytes or conjunctival fibroblast surrounding the injury site. 1 The epithelial healing process requires the coordination of several growth factors and cytokines that mediate the interactions between corneal epithelium and stroma. In addition, epithelial and stromal cells interact with each other across the basement membrane, triggering stromal responses involving apoptosis, activation, and transdifferentiation of keratocytes into myofibroblasts. 2 However, the persistence of myofibroblastic cells after wound healing can lead to the haze or scar tissue formation, reducing the patient visual capacity.
Topical lubrication is typically first line treatment with high frequency application every 1-2 h; if the patient is concurrently using medications such as antibiotics, antivirals that may alter the corneal epithelium, withdrawal of these offending agents may allow re-epithelialization. The application of therapeutic soft contact lenses serves to protect the corneal surface from mechanical trauma to the eyelids, and the use of punctal plugs should increase the retention of natural tears that aid the healing process. Surgical interventions, such as debridement and tarsorrhaphy, are other effective treatments in most cases of ocular surface disorders refractory to medical treatment, along with amniotic membrane transplantation, which has yielded satisfactory results. [3][4][5] However, these treatments have some drawbacks such as the risk of disease transmission and the limited availability of donor tissues.
Among the newest strategies as a second line of treatment, hemoderivatives as autologous serum and platelet rich plasma have been described as useful. 6,7 Platelet rich plasmas have been employed for the last years in several ophthalmic affections with successful results; it is a safer and more effective treatment than artificial tears in severe to moderate dry eye syndrome and offers a restorative response in ocular burns and limbal transplantation procedures; also, it may be an effective and safe surgical technique with satisfactory anatomical and functional results for persistent macular hole. [8][9][10][11][12] Plasma rich in growth factors (PRGF) is an autologous platelet enriched plasma free of leukocytes, obtained from the patient's own blood that clusters a lot of bioactive proteins released by platelet alpha-granules upon activation, such as epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-b1 (TGF-b1) and platelet derived growth factor (PDGF) among others. 13 With proven efficacy in ophthalmology area, PRGF eye drops have shown better results than autologous serum in ocular surface pathologies, 14,15 avoiding the inflammatory component of leukocytes and stimulating scarless regeneration in stromal fibroblasts. [16][17][18] Several studies have found topical insulin to be an effective adjuvant therapy involved in epithelial wound healing especially in refractory cases although the mechanism of action is not fully known. 19,20 In the cornea, IGF family members have been implicated in proliferation, differentiation and migration of corneal epithelial cells; 21,22 IGF-1 and its receptors are expressed by both human corneal keratocytes and epithelial cells. Recently published research has described topical insulin, concretely 1 IU/ml, as a more effective treatment for epithelization in Persistent epithelial defects (PED) than autologous serum, suggesting its use as a first-line treatment. 23 In this study, the effects of PRGF, autologous serum and topical insulin on human corneal epithelial cells, corneal fibroblasts and conjunctival fibroblasts were observed by analyzing cell proliferation, myodifferentiation and wound closure in response to the different treatments.

PRGF and autologous serum preparations
Blood from three healthy young donors (two men and a woman) was collected after informed consent into 9-ml tubes with 3.8% (wt/v) sodium citrate or in serum separator tubes (SST TM II Advance Plus Blood collection tubes, BD Vacutainer, Vaud, SUI). The study was accomplished following the principles of the Declaration of Helsinki. Blood samples for PRGF were centrifuged at 580 g for 8 min at room temperature in an Endoret System centrifuge (BTI Biotechnology Institute, S.L., Vitoria, Alava, Spain); the whole plasma column over the buffy coat was harvested using Endoret ophthalmology kit (BTI Biotechnology Institute, S.L., Vitoria, Alava, Spain) taking care not to collect the layer containing leukocytes. Platelets and leukocytes counts were performed with a hematology analyzer (Pentra ES 60, Horiba ABX, Montpellier, France). Plasma preparations were incubated with Endoret activator (BTI Biotechnology Institute, S.L., Vitoria, Alava, Spain) at 37 C for 1 h and the released supernatants were filtered, aliquoted and stored at -80 C until use.
Blood samples for autologous serum preparations were allowed to clot at room temperature for 20 min and afterwards centrifuged for 10 min at 1000 g; serum was isolated from the red series by the separation gel, filtered and collected. For further applications, serum was diluted to 20% with sterile saline solution, aliquoted and stored at -80 C until use (termed AS).

Preparation of formulations
The insulin used in this study was Actrapid V R 100 IU/mL from Novo Nordisk, Denmark. The stock insulin solution (100 IU/mL) Actrapid V R was diluted in culture medium (FM or DMEM/F12) at a concentration of 1 unit per mL (1 IU/ml) or 0.2 units per mL (0.2 IU/ml).
TGF-b1 (Invitrogen, Massachusetts, USA) preparations were used at 2.5 ng/ml to induce differentiation to myofibroblasts in culture. This type of differentiation may occur during the ocular repair process, being an undesirable effect on ocular surface tissues whether these cells remain in the ocular tissue after complete healing because they would induce fibrotic tissue, which may limit the vision of the patient. Therefore, therapeutic preparations were tested in vitro on TGF-b1 myodifferentiated cells to assess whether they could reverse this undesirable effect.

Proliferation assays
To analyze the influence of PRGF, AS, TGF-b1 and insulin over proliferation of HConF and HK, cells were seeded at a density of 7,000 cells per cm 2 on 96-well optical bottom black plates in serum-free medium supplemented with either: (i) the culture medium alone (FM) with 0.2% FBS as a control of non-stimulation (NS) (ii) 20% (v/v) PRGF or (iii) 20% (v/v) AS of the three donors, (iv) 2.5 ng/ml TGF-b1 with 0.2% FBS, (v) 1 IU/mL insulin with 0.2% FBS and (vi) 0.2 IU/mL insulin with 0.2% FBS. In another batch of experiments, after 72 h of treatment with TGF-b1 to induce the myofibroblastic phenotype, media were discarded and HConF and HK cells were incubated with the same preparations as described above.
On the other hand, HCE cells were seeded at 20,000 cells per cm 2 on 96-well optical bottom black plates in serumfree DMEM/F12 medium supplemented with either: (i) the culture medium alone with 0.2% FBS as a control of non- After 72 h of treatment, DNA content corresponding with the final number of cells in culture was quantified using the CyQUANT V R Cell Proliferation Assay (Life Technologies, Invitrogen, Carlsbad, CA, USA). Culture treatments were removed, and wells were washed carefully with phosphate buffered saline (PBS). Then microplate was frozen at -80 C for efficient cell lysis for the CyQUANT V R assay. Samples fluorescence was measured using a fluorescence microplate reader (Synergy H multimode reader, Biotek, Vermont, USA) including a DNA standard curve.

Wound healing assay
To quantify the wound healing potential of human corneal epithelial cells in response to PRGF, AS and insulin, HCE cells were plated at a very high density on a 48-well plate with culture inserts (Ibidi, GmbH, Martinsried, Germany) placed in each well and were grown until confluence. After removing the inserts carefully, two separated cell monolayers leaving a cell-free gap of approximately 900 mm thickness were created. Several wells were kept as a starting time (0 h), fixing them with 4% formaldehyde. HCE cells were then incubated with the same treatments as in the proliferation assay (PRGF, AS and insulin, using 0.2% FBS as control) for 16 h. Then culture media were removed, and cells were fixed with 4% formaldehyde, rinsed with PBS, and incubated with Hoechst 33342 in PBS. To quantify the percentage area of wound healing, fluorescence photographs of the central part of the septum before treatment (wells for 0 h) and after treatment time were captured with a digital camera coupled to an inverted microscope (Leica DFC300 FX and Leica DM IRB, Leica Microsystems). The gap area at 0 and 16 h of treatment were measured using the Image J Software (NIH, Bethesda, Maryland, USA). The results were expressed as percentage area of wound healing respecting initial time.

Myodifferentiation reversion assay and immunofluorescence detection
To analyze the capacity of PRGF, AS and Insulin to reverse a myofibroblastic phenotype, HConF and HK fibroblasts were plated at a density of 7,000 cells per cm 2 in 48-well tissue-culture plates. Cells were pretreated for 72h with 2.5 ng/ml TGF-b1 þ 0.2% FBS; then, the medium was removed, the wells were washed with PBS and cells were incubated with 20% PRGF þ 2.5 ng/mL TGF-b1, 20% AS þ 2.5 ng/ml TGF-b1, insulin 1 IU/mL þ 0.2% FBS þ 2.5 ng/mL TGF-b1, insulin 0.2 IU/mL þ 0.2% FBS þ 2.5 ng/mL TGF-b1 using 2.5 ng/ml TGF-b1 þ 0.2% FBS as a control. Culture medium alone (FM) with 0.2% FBS was also used as a non-stimulation (NS) control, in which basal expression of alpha-smooth muscle actin (a-SMA) was detected for each fibroblast cell type. After another 72h, culture media were discarded and the wells were rinsed with PBS, fixed for 10 min in cold methanol. After that, a double immunostaining was carried out to detect Ki-67 and a-SMA. Briefly, cells were blocked with 10% FBS in PBS and incubated with mouse anti a-SMA antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted to 1:800 to detect the myofibroblast differentiation in culture and rabbit anti Ki-67 antibody (Abcam, Cambridge, UK) diluted to 1:400 to detect proliferative cells, followed by incubation with corresponding secondary antibodies (488 goat anti-mouse IgG and 594 goat anti-rabbit IgG, respectively) both diluted to 1:100. Cell nuclei were counterstained with Hoechst 33342 mounted using an anti-fade solution (Southern Biotech, Birmingham, AL, USA) and visualized and photographed under a fluorescence microscope (Leica DM IRB). For a-SMA and Ki-67 positive cell counting, three random 10x microscopic fields were photographed for each immunofluorescence staining (a-SMA, Ki-67 and Hoechst 33342) on each well and images were analyzed using the Image J software. Total cell number was obtained from Hoechst staining. Hoechst (þ) and a-SMA (þ) cells were counted as myofibroblasts and Ki-67 (þ) cells were considered as proliferating cells. Finally, the percentage of a-SMA or Ki-67 positive cells was calculated for each photographed area as follows: % of a À SMA or Ki À 67 þ ð Þ cells ¼

Number of ðþÞ cells Number of total cells ðhoescht ðþÞÞ
X 100

Statistical analysis
Data were summarized as mean ± standard deviation (SD). The statistical analysis of the results was carried out by ANOVA or Kruskal-Wallis test using SPSS software (SPSS Inc., USA). A p < 0.05 was statistically significant.

Results
The exact p-values obtained in each assay performed in the present study in comparison between the different treatments were included in different tables as Supplementary Material.

PRGF and autologous serum preparations
Platelets and leukocytes were measured in blood after collection and the mean ± SD values for the three donors were 237 ± 45 Â 10 6 /ml and 5.7 ± 0.4 Â 10 6 /ml, respectively. After centrifugation and plasma obtention the values obtained were 504 ± 74 for platelets and 0.2 ± 0.1 for leukocytes. The mean platelet enrichment of the PRGF preparations was 2.1fold over the baseline concentration of platelets in whole blood.

Proliferation assays
Proliferation of HConF and HK increased after treatment with PRGF and AS when compared with the control (NS). Moreover, treatment of both types of fibroblasts with PRGF enhanced significantly their proliferation compared with AS and with insulin (1 and 0.2 IU/ml) and TGF-b1 (p < 0.001 for all comparisons). The proliferative effect of insulin (at both concentrations) over fibroblasts was very similar to that of TGF-b1 and in fact, the levels were comparable to those in control (Figure 1(A,B)), showing an inhibitory effect of TGF-b1 and insulin on the proliferation on fibroblastic cells.
Results of the proliferation assay performed on the HConF and HK cells used in the myodifferentiation assay showed that PRGF induced the highest cell proliferation rate. Furthermore, in the case of HK, the amount of DNA (ng/ml) correlating with the final number of cells was significantly higher after treatment with PRGF þ TGF-b1 compared to AS þ TGF-b1 and to insulin and TGF-b1. Even more, the proliferation of corneal fibroblasts (HK) after insulin application (1 or 0.2 IU/ml) or after TGF-b1 (2.5 ng/ml) was significantly lower than the non-stimulating situation (NS) (p < 0.001). These results were similar for HConF cells except that autologous serum was able to induce proliferation similarly to PRGF, although at somewhat lower levels ( Figure 1(C,D)).
In the case of corneal epithelial cells, the stimulation of proliferation was very similar after PRGF or AS application and this was significantly higher than in the control group (NS). However, significant differences were observed in the proliferation rate of HCE cells after PRGF treatment compared to both insulin concentrations (1 or 0.2 IU/ml). No differences were observed between the proliferative effect of insulin and AS on HCE cells (Figure 2(A)).

Wound healing assay
Human corneal epithelial cells migration was significantly stimulated after PRGF application. The area between HCE monolayers was almost totally covered by cells after 16 h of treatment with PRGF, while in the other groups the wound area was only partially closed (Figure 2(B)). Insulin at both concentrations maintained the migratory capacity at levels like autologous serum and slightly higher than the control treatment. Figure 2(C) shows immunofluorescence Hoechst representative images of HCE cells at the initial state (starting time) and after 16 h of wound closure and highlights the potent stimulatory effect of PRGF over the treated cells. Results from the in vitro wound closure assay revealed that treatment with PRGF treatment achieved an 80% closure rate compared to baseline while in the autologous serum, insulin 1 and 0.2 IU/ml and control groups it was only partially closed with percentages of 55, 50, 54, and 43%, respectively. The migration time was stopped at 16 h to detect differences between groups before the gap was completely closed.

Myodifferentiation reversion assay and immunofluorescence detection
Results showed that percentage of HConF and HK a-SMA positive cells was significantly lower in PRGF treated cells than in AS treated cells and much lower than for insulin and TGF-b1. These results correspond to a higher presence of myofibroblasts when PRGF was not applied (Figures 3(A)  and 4(A)). In parallel, the proliferation of both cell types measured by the percentage of Ki-67 expression with respect to the total number of cells was higher in fibroblasts treated with PRGF than with AS or insulin or TGF-b1 (Figures 3(B) and 4(B)). Thus, an elevated level of a-SMA together with low Ki-67 expression means that the fibroblast is differentiating to a myofibroblastic phenotype rather than proliferating.
Immunofluorescence detection of a-SMA revealed that PRGF inhibited the differentiation of the different fibroblast populations into myofibroblasts (Figures 3(C) and 4(C)). Indeed, PRGF was able to maintain HConF and HK proliferative activity significantly while reducing myodifferentiation expression; this was not achieved with AS, let alone insulin which gave virtually the same results as TGF-b1 in terms of a-SMA and Ki-67 expression.

Discussion
The present experimental study shows that plasma rich in growth factors eye drops (PRGF) exert a similar proliferative effect on corneal epithelial cells (HCE) to autologous serum, but it was significantly increased after PRGF treatment compared to insulin. However, wound healing area was significantly reduced after PRGF treatment in comparison with AS or insulin. As suggested in previous studies, these differences between PRGF and AS may be due to the high EGF content of PRGF samples with respect to AS. 16 On the other hand, the differences obtained between PRGF and insulin could be explained by the content of several growth factors in PRGF which encourage chemotaxis in corneal epithelial cells, such as EGF, IGF, hepatocyte growth factor (HGF) and nerve growth factor (NGF) compared to insulin, whose action on corneal epithelial cells is suggested to be through binding to IGF receptors. 20,[24][25][26] Yet, although previous study showed significant differences in corneal re-epithelialization time between AS and insulin, 23 no differences in the wound healing area were found between AS and insulin (0.2 or 1 IU/mL) in the present study. For clinical studies, the insulin stock at 100 IU/mL was diluted in a polyethylene glycol (PEG) and polypropylene glycol (PG) solution to obtain an insulin dilution of 1 IU/mL. 20,23,27 The presence of PEG and PG increases the viscosity of insulin eye drops dilating their bioavailability on the ocular surface, and consequently, increasing their action on corneal epithelial cells. 28 Therefore, a greater availability of insulin by ocular surface tissues could explain the significant differences obtained in clinical studies between insulin and AS in corneal re-epithelialization time. In this experimental study, the proliferative potential of two different blood derived products such as AS and PRGF eye drops was compared with two concentrations of insulin (0.2 and 1 IU/mL) in two types of primary fibroblast cells (keratocyte and conjunctival fibroblast cells). Additionally, the antifibrotic capacity of the different treatments was also evaluated in HK and HConF cells after their differentiation into myofibroblasts using TGF-b1. The results observed in the present study showed that PRGF treatment significantly increased the number of HConF and HK cells compared with AS and insulin at 1 or 0.2 IU/mL. Likewise, except for AS-treated HConF cells, these differences were maintained even in cells that had been myodifferentiated. Moreover, AS significantly induced an increase in the number of HConF and HK cells with respect to both insulin concentrations also in those cells that were previously differentiated to myofibroblasts. On the other hand, no proliferative effect was observed in HConF and HK cells treated with insulin at both concentrations, showing a similar cell number to that of the non-stimulated or TGF-b1 treated group. These results correlate with the expression of Ki-67, a proliferative marker, in HConF and HK cells incubated with the different treatments after myofibroblast differentiation. Furthermore, PRGF significantly reduces a-SMA expression in myodifferentiated HConF and HK cells compared to AS and insulin, suggesting that PRGF exerts significantly greater inhibition on TGF-b1-induced myofibroblasts in conjunctival fibroblasts and corneal stromal keratocytes regarding to AS and insulin, which show a reduced ability to protect fibroblast cells against myofibroblast transformation. These results suggest that PRGF significantly enhances ocular surface tissue regeneration compared to SA or insulin because PRGF enhances fibroblast cell proliferation favoring the repopulation of injured tissues while reducing fibrosis formation.
The results in primary fibroblast cells (HConF and HK cells) obtained in the present study between PRGF and AS corroborate the results of a previous study that showed that PRGF induces a greater proliferative effect than AS on fibroblast cell types, and PRGF also induces a greater protective and inhibitory effect on TGF-b1-induced myofibroblast transformation with respect to AS. 16 Although the concentration of TGF-b1 in PRGF is 4-folds higher than the levels observed in 20% AS 16 and tears, 18 several studies have demonstrated that PRGF reduces a-SMA expression and proteins related to its expression in TGF-b1-treated ocular surface fibroblasts regarding 20% AS and even compared to 100% AS. 16  Although the mechanism by which insulin acts on ocular surface cells is not well known, it has been suggested that it acts through binding to IGF receptors. 20 It has been suggested that corneal keratocytes differentiated from mesenchymal stem cells express the IGF-I receptor; 22 however, the scarce studies published in the literature on IGF receptor expression by corneal keratocytes showed that this cell type express the IGF-2 receptor (IGF2R). 31 IGF2R is a large transmembrane glycoprotein highly conserved in all vertebrates, and although its function is not well known, several studies suggest that it is a multifunctional receptor, but its main activity is to limit cell growth. 32 This could explain why insulin-treated fibroblast cells grow similarly to nonstimulated cells and has no effect on myofibroblast cell reversion.
In summary, the results obtained in the present study show that PRGF significantly reduces the re-epithelialization time of corneal epithelial cells compared to insulin. Furthermore, PRGF eye drops induce an increase in the proliferation rate of ocular surface cells of fibroblastic origin while reducing the presence of myofibroblastic cells in comparison with insulin and autologous serum. Although this is a preliminary study, the results obtained in the present study suggest that PRGF may improve the treatment of ocular surface wound healing minimizing the scar formation compared to insulin.