Comparison of corneal epitheliotrophic capacities among human platelet lysates and other blood derivatives

Abstract

Purpose: To evaluate the corneal epitheliotropic abilities of two commercialized human platelet lysates (HPLs) and to compare the results with other blood derivatives, including human peripheral serum (HPS) and bovine fetal serum (FBS).

Methods: In vitro, human corneal epithelial cells were incubated in various concentrations (0%, 3%, 5% and 10%) of blood derivatives. Two commercialized HPLs, including UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were tested and compared with HPS and FBS. Scratch-induced directional wounding assay was performed to evaluate cellular migration. MTS assay was used to evaluate cellular proliferation. Cellular differentiation was examined by scanning electron microscopy, inverted microscopy and transepithelial electrical resistance. Sprague-Dawley rats were used to evaluate the effects of the blood derivatives on corneal epithelial wound healing in vivo. Different blood derivatives were applied topically every 2 hours for 2 days after corneal epithelial debridement. The concentrations of epidermal growth factor (EGF), transforming growth factor -β1 (TGF-β1), fibronectin, platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, and hyaluronic acid in different blood derivatives were evaluated by enzyme-linked immunosorbent assay (ELISA).

Results: In vitro experiments demonstrated statistically comparable epitheliotropic characteristics in cellular proliferation, migration, and differentiation for the two commercialized HPLs compared to FBS and HPS. Cells cultured without any serum were used as control group. The epitheliotropic capacities were statistically higher in the two commercialized HPLs compared to the control group (p<0.05). Among the different concentrations of blood derivatives, the preparations with 3% yielded better outcomes compared to 5% and 10%. In rats, HPLs also caused improved but not statistically significant wound healing compared to HPS. All the blood derivatives had better wound healing ratios than the control group (p<0.05). In the quantification of epitheliotropic factors, UltraGRO and PLTMax had significantly higher levels of EGF, TGF- β1, fibronectin than human peripheral serum (p<0.05).

Conclusions: Both commercialized HPLs showed comparable corneal epitheliotropic abilities and wound healing rates compared to HPS and FBS in the in vivo and in vitro studies. Our results suggest that HPLs may have the potential to replace HPS in the treatment of corneal epithelial problems.

Fig 1. Cell migration: Scratch-induced directional wounding assay at 16 hours after wounding.

(A) 10% preparations of blood derivatives demonstrated poorer response compared to 3% and 5% preparations. There was no significant difference among the 4 blood derivatives in 3% and 5% preparations. * indicated p <0.05. ** indicated p <0.01 compared to the control group that was without blood derivatives in culture media. (B) Representative picture of the effects of different blood derivatives on epithelial scratch wound healing at 16 hours after wounding. FBS: fetal bovine serum, HPS: human peripheral serum. UltraGRO: Human platelet lysate from Helios pharmaceutical. PLTMax: Human platelet lysate from Mill Creek pharmaceutical.

Fig 2. Cell proliferation: The effects of different blood derivatives on cellular proliferation with MTS assay.

At 24 hours and 48 hours, corneal epithelial cells incubated with fetal bovine serum had significantly higher proliferative responses than those incubated in human peripheral serum, UltraGRO human platelet lysate (HPL), and PLTMax HPL. At 72 hours, there was no difference among these 4 products in 3% and 5% preparations. At 24, 48, and 72 hours, the proliferative responses showed no statistical difference between 3% and 5% blood derivative preparations. However, 10% preparation demonstrated poorer proliferative response compared to 3% and 5% preparations at all time points. FBS: fetal bovine serum, HPS: human peripheral serum. UltraGRO: Human platelet lysate from Helios pharmaceutical. PLTMax: Human platelet lysate from Mill Creek pharmaceutical. * indicated p <0.05. ** indicated p <0.01 compared to the control group that was without blood derivative in culture media.

Fig 3. Cell differentiation: Inverted microscopy and scanning electron microscopy.

Morphologies of human corneal epithelial cell (HCEC) lines cultivated for 48 hours with different blood derivatives, including (A) no serum, (B) human peripheral serum (HPS), (C) UltraGRO human platelet lysate (HPL), and (D) PLTMax HPL. Under inverted microscopy, cells incubated without serum formed a coherent monolayer of irregularly shaped cells (A) while cells cultivated in HPS, UltraGro, and PLTMax showed regular, polygonal flat cells (B-D). Under SEM, cells cultivated without serum showed increased cell-to-cell junctions without the formation of microvilli on the cell surface (A). The white arrow indicated the cell-to-cell junction and the white arrow heads represented the microvilli on the cell surface. Exfoliation of the cells from the culture dish was suspected in cells cultivated without blood derivatives. B-D) Cells cultivated in HPS, UltraGro, and PLTMax demonstrated tight cell-to-cell junction with upright microvilli homogenously and densely distributed at the cellular surface. Original magnifications of the inverted microscopy: 100X and SEM 1250X. UltraGRO: Human platelet lysate from Helios pharmaceutical. PLTMax: Human platelet lysate from Mill Creek pharmaecutical.

Fig 4. Cell differentiation: Measurement of trans-epithelial electrical resistance (TEER).

The effects of different blood derivatives on trans-epithelial electrical resistance. Cells incubated with fetal bovine serum (FBS), human peripheral serum (HPS), UltraGRO human platelet lysate (HPL), and PLTMax HPL demonstrated significantly higher TEER values compared to the control group. However, there was no significant differences among FBS, HPS, UltraGRO, and PLTMax on day 3 after the cells reached confluency. UltraGRO: Human platelet lysate from Helios pharmaceutical. PLTMax: Human platelet lysate from Mill Creek pharmaceutical. * indicated p <0.01. ** indicated p <0.01 compared to the control group that was without any blood derivative.

Fig 5. Rat model of corneal epithelial wound healing.

(A) In vivo rat corneal epithelial wound healing after epithelial debridement and topical treatment with 20% of human peripheral serum (HPS) and the 2 different human platelet lysates (HPLs). There were significantly better wound healing abilities in HPS and the 2 HPL groups compared to the control group at 24 hours after wounding. (B) Representative pictures of corneal epithelial defects at 12, 24 and 48 hours after surgery in rat eyes that received epithelial debridement and different treatments. UltraGRO: Human platelet lysate from Helios pharmaceutical. PLTMax: Human platelet lysate from Mill Creek pharmaceutical. * indicated p<0.05 which was compared to the control group without any blood derivative.