The corneal epitheliotrophic abilities of lyophilized powder form human platelet lysates

Abstract

Purpose: To evaluate whether lyophilized human platelet lysate (HPL) powder can preserve the growth factor concentrations and epitheliotrophic properties of liquid HPL, and potentially be used as a clinically-friendly treatment option.

Methods: Two commercialized liquid HPLs, UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were obtained and converted to lyophilized powder. After redissolution, lyophilized powder HPLs were compared with liquid HPLs, as well as human peripheral serum (HPS) and fetal bovine serum (FBS) in liquid or redissolved lyophilized powder forms. Concentrations of epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-AB (PDGF-AB) and platelet-derived growth factor-BB (PDGF-BB) were evaluated by enzyme-linked immunosorbent assay (ELISA). Human corneal epithelial cell line was incubated with the blood derivatives and evaluated for cell migration with scratch-induced directional wounding and proliferation with MTS assays. Cell differentiation was examined by transepithelial electrical resistance (TEER). Fluorescein staining and in vivo confocal microscopy were used to evaluate in vivo corneal epithelial wound healing in Sprague-Dawley rats that underwent corneal debridement and topical application of liquid and redissolved powder HPLs.

Results: Liquid form and redissolved lyophilized powder form HPLs had similar concentrations of EGF, TGF-β1, PDGF-AB and PDGF-BB. In vitro experiments on cell migration, proliferation and differentiation and rat models on wound healing demonstrated no significant difference between the liquid and redissolved lyophilized powder forms for HPLs, HPS and FBS. In vivo confocal microscopy revealed similar wound healing process at different layers of cornea after corneal epithelial debridement between liquid form and redissolved lyophilized power form of HPLs.

Conclusions: The redissolved lyophilized powder form of both commercialized HPLs showed similar growth factor concentrations and corneal epitheliotrophic abilities compared to the liquid form. Results suggest that the properties of liquid HPLs can be retained despite lyophilization and that lyophilized HPLs can be a treatment option for corneal epithelial disorders.

Fig 1. Appearance of various forms of HPS.

(A) liquid form. (B) lyophilized powder form. (C) redissolved lyophilized powder form in plastic tube. (D) redissolved lyophilized powder form in plastic well of a 12-well plate (volume of 1ml). Redissolved lyophilized powder form HPS showed clear, faint yellow color with satisfactory transparency.

Fig 2. Comparison of liquid and redissolved lyophilized powder forms of HPS and HPLs.

Control column on the left shows the NTUH (National Taiwan University Hospital) logo, plastic well, and plastic well containing PBS. HPS and HPLs (UltraGRO and PLTMax) shown on the right appear as clear yellow solutions with similar levels of transparency between the the liquid and redissolved lyophilized powder forms.

Fig 3. Quantification of epitheliotrophic factors in liquid and redissolved lyophilized powder forms of HPLs with the ELISA assay.

Concentrations of (A) EGF, (B) PDGF-AB, (C) PDGF-BB and (D) TGF-β1 were measured in UltraGRO and PLTMax. There were no statistically significant differences in the concentration levels between liquid and redissolved powder forms (p>0.05). Error bars indicate SD.

Fig 4. Scratch-induced directional wounding assay at 16 hours.

(A) HCECs were cultured in 5% blood derivatives (FBS, HPS, UltraGRO and PLTMax) in liquid or redissolved lyophilized powder forms, and tested for wound-healing after scratching. All blood derivatives had increased wound-healing ratios compared to the control (serum-free) at 16 hours (p<0.01). There were no statistically significant differences in wound healing ratios between liquid and redissolved powder forms (p>0.05). **p<0.01 compared to the control. Error bars indicate SD. (B) Representative images from inverted microscopy that was done to evaluate wound healing in HCECs cells cultured in 5% blood derivatives (FBS, HPS, UltraGRO and PLTMax) in liquid and redissolved lyophilized powder forms. Control represents the original scraping area at 0 hours after injury.

Fig 5. MTS assay to evaluate cell proliferation.

HCEC were cultured in blood derivatives (FBS, HPS, UltraGRO and PLTMax) at 3%, 5% and 10% concentrations and tested with the MTS assay at 24, 48, and 72 hours. Compared to the control (no serum), cell incubated in blood derivatives resulted in increased proliferation at 48 and 72 hours. Liquid and redissolved lyophilized powder forms produced similar results at almost all concentrations and time points (p>0.05). *p<0.05 compared to the control.**p<0.01 compared to the control. Error bars indicate SD.

Fig 6. TEER assay to evaluate cell differentiation and function.

HCEC were cultured in 5% blood derivatives (liquid and redissolved lyophilized powder forms of FBS, HPS, UltraGRO and PLTMax) and measured for TEER values on day 3. Compared to the control (no serum), cell incubated in blood derivatives produced increased TEER values. Liquid and redissolved lyophilized powder forms gave similar results (p>0.05). **p<0.01 compared to the control. Error bars indicate SD.

Fig 7. Corneal epithelial wound healing in rats.

Rats underwent corneal epithelial debridement and topical applicaton of liquid or redissolved powder forms of blood derivatives. Corneal epithelial defects were stained with fluorescein and photographed at 0, 12, 24 and 48 hours to determine wound healing ratios. Compared to the control (no topical treatment), rats treated with blood derivatives had greater wound healing ratios at 24 hours. Effects were similar for liquid and redissolved powder forms. All rats had fully healed corneal epithelium at 48 hours. *p<0.05 compared to the control. **p<0.01 compared to the control. Error bars indicate SD.

Fig 8. Cell morphologies during corneal epithelial wound healing in rats.

Images were taken at 48 hours with a HR3 confocal microscope to view corneal epithelium for rats that underwent no corneal epithelial debridement (normal cornea), rats that underwent debridement but no treatment (control group), and rats that underwent debridement and treatment with UltraGRO (liquid form and powder form). Corneal epithelial debridement treated with UltraGRO had healed apical and basal epithelia similar to those of normal cornea, while the control had dry apical squamous cells and WBC infiltrates in the basal epithelium. The superficial stroma was not damaged, and appeared similar across the different groups. Image dimensions of 0.4mm x 0.4mm.