Ana Cristina Riestra

Autologous serum and plasma rich in growth factors in ophthalmology: preclinical and clinical studies.

The use of blood derivatives represents an alternative therapeutic approach that is gaining interest in regenerative medicine due to its potential to stimulate and accelerate tissue healing. Autologous serum eye drops and platelet‐enriched plasma eye drops are being used in the treatment of different ophthalmological disorders. In this review, we summarize the different blood‐derived formulations used in the treatment and care of ocular surface disorders. The biological basis and use of autologous serum and plasma rich in growth factors are deeply evaluated as well as the challenges to be addressed in the future in this new generation of blood‐derived therapies.

Abnormal activity of corneal cold thermoreceptors underlies the unpleasant sensations in dry eye disease

Abstract Dry eye disease (DED) affects >10% of the population worldwide, and it provokes an unpleasant sensation of ocular dryness, whose underlying neural mechanisms remain unknown. Removal of the main lachrymal gland in guinea pigs caused long-term reduction of basal tearing accompanied by changes in the architecture and density of subbasal corneal nerves and epithelial terminals. After 4 weeks, ongoing impulse activity and responses to cooling of corneal cold thermoreceptor endings were enhanced. Menthol (200 &mgr;M) first excited and then inactivated this augmented spontaneous and cold-evoked activity. Comparatively, corneal polymodal nociceptors of tear-deficient eyes remained silent and exhibited only a mild sensitization to acidic stimulation, whereas mechanonociceptors were not affected. Dryness-induced changes in peripheral cold thermoreceptor responsiveness developed in parallel with a progressive excitability enhancement of corneal cold trigeminal ganglion neurons, primarily due to an increase of sodium currents and a decrease of potassium currents. In corneal polymodal nociceptor neurons, sodium currents were enhanced whereas potassium currents remain unaltered. In healthy humans, exposure of the eye surface to menthol vapors or to cold air currents evoked unpleasant sensations accompanied by increased blinking frequency that we attributed to cold thermoreceptor stimulation. Notably, stimulation with menthol reduced the ongoing background discomfort of patients with DED, conceivably due to use-dependent inactivation of cold thermoreceptors. Together, these data indicate that cold thermoreceptors contribute importantly to the detection and signaling of ocular surface wetness, and develop under chronic eye dryness conditions an injury-evoked neuropathic firing that seems to underlie the unpleasant sensations experienced by patients with DED.

Plasma rich in growth factors (PRGF) eye drops stimulates scarless regeneration compared to autologous serum in the ocular surface stromal fibroblasts

Autologous serum (AS) eye drops was the first blood-derived product used for the treatment of corneal pathologies but nowadays PRGF arises as a novel interesting alternative to this type of diseases. The purpose of this study was to evaluate and compare the biological outcomes of autologous serum eye drops or Plasma rich in growth factors (PRGF) eye drops on corneal stromal keratocytes (HK) and conjunctival fibroblasts (HConF). To address this, blood from healthy donors was collected and processed to obtain autologous serum (AS) eye drops and plasma rich in growth factors (PRGF) eye drops. Blood-derivates were aliquoted and stored at -80°C until use. PDGF-AB, VEGF, EGF, FGFb and TGF-β1 were quantified. The potential of PRGF and AS in promoting wound healing was evaluated by means of proliferation and migration assays in HK and HConF. Fibroblast cells were induced to myofibroblast differentiation after treatment with 2.5ng/mL of TGF-β1. The capability of PRGF and AS to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results showed significant higher levels of all growth factors analyzed in PRGF eye drops compared to AS. Moreover, PRGF eye drops enhanced significantly the biological outcomes of both HK and HConF, and reduced TGF-β1-induced myofibroblast differentiation in contrast to autologous serum eye drops (AS). In summary, these results suggest that PRGF exerts enhanced biological outcomes than AS. PRGF may improve the treatment of ocular surface wound healing minimizing the scar formation compared to AS. Results obtained herein suggest that PRGF protects and reverses the myofibroblast phenotype while promotes cell proliferation and migration.

Safety and Efficacy of Autologous Plasma Rich in Growth Factors Eye Drops for the Treatment of Evaporative Dry Eye

Purpose: To provide preliminary information about the safety and efficacy of plasma rich in growth factors (PRGF) in the treatment of evaporative dry eye (EDE) and analyze the influence of certain variables on treatment outcomes. Methods: This retrospective study included patients with EDE treated between January 2011 and December 2013. Outcome measures including signs and symptoms of the disease were evaluated before and after treatment. The influence of some of the patients clinical variables on results was assessed. Safety assessment was also performed. Results: Eighty-three patients with a total of 156 eyes were evaluated. Statistically significant reductions in the Ocular Surface Disease Index (38.2%), best-corrected visual acuity (27.4%), Visual Analogue Scale scores for frequency (32%) and severity (34%), and a significant improvement in the Schirmer test scores (177.5%) were observed (p < 0.05). The results were stratified according to the identified potential effect modifiers. Only four adverse events were reported. All of them were mild and resolved within a few days. Conclusions: Results suggest that PRGF eye drops could be a safe and effective treatment in reducing EDE signs and symptoms. When treating patients, the possible influence of some clinical variables must be taken into account.

Autologous Plasma Rich in Growth Factors Eyedrops in Refractory Cases of Ocular Surface Disorders

Purpose: Preliminary information about the safety and efficacy of plasma rich in growth factors (PRGF) eyedrops in the treatment of refractory cases of diverse ocular surface disorders (OSDs) is presented here. Material and Methods: This retrospective cohort study included cases with OSDs refractory to previous treatment with conventional treatments or autologous serum or cyclosporine, and treated with PRGF eyedrops. The signs and symptoms of ocular surface disorders [using the ocular surface disease index (OSDI), best-corrected visual acuity (BCVA), visual analog scale (VAS) frequency and VAS severity] were evaluated before and after treatment with PRGF. A safety assessment was also performed reporting all adverse events or complications. Results: Forty-one patients with a total of 80 treated eyes were evaluated. Statistically significant reductions in the OSDI scale (39.27%), VAS frequency (38.9%) and VAS severity (40.3%), and a significant improvement in BCVA (54.86%) were all observed (p < 0.05). The results were stratified according to the identified potential effect modifiers. There were only two adverse events (eye redness and eyelid inflammation), which were reported as mild and resolved in a few days. Conclusions: PRGF eyedrops could be a safe and effective treatment option for refractory cases of OSDs. When treating patients the possible influence on the results of some clinical variables must be taken into account.

Polymeric nanocapsules: a potential new therapy for corneal wound healing

Corneal injuries are one of the most frequently observed ocular diseases, leading to permanent damage and impaired vision if they are not treated properly. In this sense, adequate wound healing after injury is critical for keeping the integrity and structure of the cornea. The goal of this work was to assess the potential of polymeric nanocapsules, either unloaded or loaded with cyclosporine A or vitamin A, alone or in combination with mitomycin C, for the treatment of corneal injuries induced by photorefractive keratectomy surgery. The biopolymers selected for the formation of the nanocapsules were polyarginine and protamine, which are known for their penetration enhancement effect. The results showed that, following topical instillation to a mouse model of corneal injury, all the nanocapsule formulations, either unloaded or loaded with cyclosporine A or vitamin A, were able to stimulate corneal wound healing. In addition, the healing rate observed for the combination of unloaded protamine nanocapsules with mitomycin C was comparable to the one observed for the positive control Cacicol®, a biopolymer known as a corneal wound healing enhancer. Regarding the corneal opacity, the initial grade of corneal haze (>3) induced by the photorefractive keratectomy was more rapidly reduced in the case of the positive control, Cacicol®, than in corneas treated with the nanocapsules. In conclusion, this work shows that drug-free arginine-rich (polyarginine, protamine) nanocapsules exhibit a positive behavior with regard to their potential use for corneal wound healing.

Preservation of Biological Activity of Plasma and Platelet-Derived Eye Drops After Their Different Time and Temperature Conditions of Storage.

Purpose: To analyze whether plasma rich in growth factors (PRGF) eye drops preserve their biological characteristics and activity after storage for 3 and 6 months at −20°C, at 4°C, and at room temperature for 72 hours, compared with fresh samples (t0). Methods: Blood from 6 healthy donors was harvested and centrifuged to obtain PRGF free of leukocytes. Resulting PRGF eye drops were stored for 3 and 6 months at −20°C. At each time, 2 aliquots were maintained at room temperature or at 4°C for 72 hours. Platelet-derived growth factor-AB, transforming growth factor-&bgr;1, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor-1, angiopoietin-1, and thrombospondin-1 were quantified at each time and temperature of storage. Also, the effect of PRGF eye drops on proliferation of primary human keratocytes was evaluated. Results: All the analyzed growth factor levels remained constant at each time and storage condition. No differences were observed in the proliferative activity of keratocytes after treatment with PRGF eye drops at any studied time or temperature. Finally, there was no microbial contamination in any of the PRGF eye drops. Conclusions: The preservation of the PRGF eye drops at −20°C for up to 3 and 6 months does not mean reduction of the main growth factors and proteins implicated in ocular surface wound healing. Eye drop characteristics and in vitro biological activity were not affected by their usage and conservation for 72 hours at 4°C or at room temperature.

Silk Fibroin Films for Corneal Endothelial Regeneration: Transplant in a Rabbit Descemet Membrane Endothelial Keratoplasty

Purpose Develop a silk fibroin (SF)-based artificial endothelial graft for its use in a rabbit Descemet membrane endothelial keratoplasty (DMEK). Methods Human and rabbit artificial corneal endothelial grafts were developed through the culture of human and rabbit corneal endothelial cells (CECs) on SF films. Rabbit artificial SF endothelial grafts were transplanted in a DMEK surgery into a rabbit in vivo model. Results SF artificial endothelial grafts showed the characteristic endothelial markers: zonula occludens (ZO-1) and Na+/K+ ATPase. In a rabbit model of DMEK surgery, SF artificial endothelial graft restored the corneal transparency and thickness at 6 week of follow-up. Anterior segment optical coherence tomography revealed the SF graft as a fully integrated component in the corneal tissue, displaying a similar corneal thickness and endothelial cell count when compared with its healthy contralateral cornea. Histologic analysis showed that the SF artificial endothelial graft was attached and integrated on the surface of the corneal stroma without a significant inflammatory reaction, and rabbit CECs consisted in a monolayer that showed their characteristic markers ZO-1 and Na+/K+ ATPase, suggesting proper intercellular junctions and cellular pump function. Conclusions We have developed SF films with biological properties that supported the growth of rabbit and human CECs, which showed normal morphology and characteristic markers; and with mechanical properties that allowed its use in a DMEK surgery, proving its in vivo functionality in a rabbit model of endothelial dysfunction.

PRGF exerts more potent proliferative and anti-inflammatory effects than autologous serum on a cell culture inflammatory model.

Ocular graft versus host disease (oGVHD) is part of a systemic inflammatory disease that usually affects ocular surface tissues manifesting as a dry eye syndrome. Current treatments provide unsatisfactory results. Blood-derived products, like plasma rich in growth factors (PRGF) emerge as a potential therapy for this disease. The purpose of this study was to evaluate the tissue regeneration and anti-inflammatory capability of PRGF, an autologous platelet enriched plasma eye-drop, compared to autologous serum (AS) obtained from oGVHD patients on ocular surface cells cultured in a pro-inflammatory environment. PRGF and AS were obtained from four GVHD patients. Cell proliferation and inflammation markers, intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2), were measured in corneal and conjunctival fibroblastic cells cultured under pro-inflammatory conditions and after treatment with PRGF or AS eye drops. Moreover, cell proliferation increased after treatment with PRGF and AS, though this enhancement in the case of keratocytes was significantly higher with PRGF. PRGF eye drops showed a significant reduction of both inflammatory markers with respect to the initial inflammatory situation and to the AS treatment. Our results concluded that PRGF exerts more potent regenerative and anti-inflammatory effects than autologous serum on ocular surface fibroblasts treated with pro-inflammatory IL-1β and TNFα.