Stephanie Watson

A contact lens-based technique for expansion and transplantation of autologous epithelial progenitors for ocular surface reconstruction.

Background. A healthy cornea is reliant on a distinct population of stem cells (SC) that replace damaged or aging epithelium throughout life. Depletion of the SC pool or damage to the niche can result in a blinding and painful condition known as limbal-SC deficiency (LSCD). Although current treatment strategies for reconstituting the ocular surface for patients suffering LSCD are promising, they are complicated by transferring autologous or allogeneic progenitors in the presence of animal, human, and synthetic products. We report on the safe and efficacy of a unique autologous SC transfer technique that utilizes an Food and Drug Administration-approved contact lens (CL) as the SC substrate and carrier for patients with LSCD. Methods. Three patients with LSCD due to aniridia (n=1) and posttreatment for recurrent ocular surface melanoma (n=2) were included. Limbal (n=2) or conjunctival biopsies (n=1) were harvested and progenitors expanded ex vivo on therapeutic CLs in the presence of autologous serum. Cell-laden CLs were transferred to the patients corneal surface and clinical outcome measures were recorded (follow-up range, 8–13 months). Results. A stable transparent corneal epithelium was restored in each patient. There was no recurrence of conjunctivalization or corneal vascularization, and a significant improvement in symptom score occurred in all patients. Best-corrected visual acuity was increased in all eyes after the procedure. Conclusion. Ex vivo expansion of ocular surface epithelium in the presence of autologous serum and transplantation with the aid of a soft CLs is a promising new technique capable of achieving ocular surface rehabilitation.

Use of Intraoperative Fourier-Domain Anterior Segment Optical Coherence Tomography During Descemet Stripping Endothelial Keratoplasty

PURPOSE To evaluate the intraoperative use of handheld Fourier-domain optical coherence tomography (OCT) during Descemet stripping automated endothelial keratoplasty (DSAEK) to assess the donor-host interface. DESIGN Prospective, observational case series. METHODS Six patients undergoing DSAEK surgery were included. OCT scans of the cornea were performed intraoperatively after insertion of the donor disc, after instillation of air in the anterior chamber beneath the disc, after vent incisions in the host cornea in each quadrant, following air-fluid exchange at the end of operation, and on day 1 after surgery. The central 3 mm of each cornea was scanned. The broadest gap between donor and host cornea (interface space) was measured. RESULTS Adequate readings could be obtained from all patients without any complications. In 2 patients there was a decrease in the width of the interface space after each surgical step documented by the OCT scans. At the end of their operation, no interface space was detectable. In 2 patients, interface space disappeared after the vent incisions and did not reappear during the further course of the surgery. In further 2 patients the separation between the host and donor was still detectable at the end of the operation. All patients had no detectable interface gap on day 1. CONCLUSIONS Handheld anterior segment OCT can be used to assess the host-donor interface in lamellar corneal transplantation surgery. Donor adherence can occur in spite of residual interface space at the end of surgery. Further studies should be conducted to answer the question of which surgical steps are useful in assisting with donor adhesion.

Diversity of microbial species implicated in keratitis: a review.

Background: Microbial keratitis is an infectious disease of the cornea characterised by inflammation and is considered an ophthalmic emergency requiring immediate attention. While a variety of pathogenic microbes associated with microbial keratitis have been identified, a comprehensive review identifying the diversity of species has not been completed. Methods: A search of peer-reviewed publications including case reports and research articles reporting microorganims implicated in keratitis was conducted. Search engines including PubMed, Scopus and Web of Science with years ranging from 1950-2012 were used. Results: 232 different species from 142 genera, representing 80 families were found to be implicated in microbial keratitis. Fungi exhibited the largest diversity with 144 species from 92 genera. In comparison, 77 species of bacteria from 42 genera, 12 species of protozoa from 4 genera and 4 types of virus were identified as the infectious agents. A comparison of their aetiologies shows reports of similarities between genera. Conclusions: The diversity of microbial species implicated in keratitis has not previously been reported and is considerably greater than suggested by incidence studies. Effective treatment is heavily reliant upon correct identification of the responsible microorganisms. Species identification, the risk factors associated with, and pathogenesis of microbial keratitis will allow the development of improved therapies. This review provides a resource for clinicians and researchers to assist in identification and readily source treatment information.

Legeais BioKpro III keratoprosthesis implantation: long term results in seven patients

Aims: The long term results of the Legeais BioKpro III keratoprosthesis are presented for seven patients with severe corneal scarring. Methods: The study took place at Moorfields Eye Hospital, London. Patients had either end stage ocular surface disease or corneal opacification after multiple failed graft surgery, with the potential for significant visual improvement. After insertion the device was covered with a conjunctival flap or buccal mucous membrane graft, which was later opened to expose the optic. The outcome measures were vision, complications, and retention of the device. Results: The BioKpro III was inserted into seven patients with severe corneal scarring: ocular cicatricial pemphigoid, measles keratitis, thermal injury, Stevens-Johnson syndrome, aniridia, chemical injury, and congenital rubella. The follow up was 18–48 months. The keratoprosthesis failed in six, because of extrusion occurring 2–28 months postoperatively. Retroprosthetic membranes occurred in three patients, and endophthalmitis in one. Vision improved from hand movements to 6/12 in the only patient who retained the KPro; however he was troubled by mucus accumulation on the optic. Conclusions: The one success has been in a patient with thermal burns. The remaining results have been poor, with the KPro extruding in six of the seven patients.

Vitronectin: a matrix support factor for human limbal epithelial progenitor cells.

PURPOSE The authors recently developed a therapeutic technique for patients with limbal stem cell deficiency by harvesting ocular surface stem cells (SCs), expanding them on therapeutic contact lenses (CLs), and applying them to diseased corneas. The present study determined the proteins that bind to CLs and whether such factors, along with transplanted cells, are critical determinants for corneal rehabilitation using this method. METHODS Therapeutic CLs were exposed to human serum, and adherent proteins were analyzed by proteomics. The distribution of vitronectin (VN) on the ocular surface was determined with specific antibodies. Cadaveric human corneas were chemically wounded, and cell transfer by CLs was assessed in organ culture. RESULTS VN was identified as a serum factor that binds and desorbs from CLs. VN localized to the limbal and basement membranes (BM) of other SC-harboring organs. Clonogenic assays demonstrated higher colony-forming efficiency on VN compared with uncoated surfaces. Cell transfer from CLs was achieved through in vitro models and was abrogated by RGD peptides and inhibitory antibodies to VN and its receptor. CONCLUSIONS Identification of VN within the limbal BM, its effect on limbal SC activity, and the discovery of this factor on serum-exposed CL polymers implies a role in supporting progenitor cells and facilitating corneal regeneration.

Retinoic acid and the ocular surface.

Retinoic acid is known to improve cutaneous wound healing and, in recent years, its application in ophthalmology has been investigated. This review looks at the role of retinoic acid on the ocular surface. Retinoic acid can be produced synthetically, and its mechanism of action includes both nuclear and non-nuclear receptor mediated pathways. It has been shown to improve full and partial thickness corneal lacerations as well as corneal epithelial defects. Retinoic acid plays a critical role in cell differentiation at the cornea, conjunctiva, and limbus, and may have an anti-tumor role. Its positive effect is only achieved at the correct concentration, however; excess concentrations of retinoic acid have a deleterious effect. The main limiting factor of retinoic acid use is its detrimental effect on meibomian glands, resulting in cell death, atrophy of acini, hyposecretion of oils, and altered gene expression, eventually resulting in dry eye symptoms. This effect is reversible on discontinuation of the drug.

Clinical outcomes of xeno-free expansion and transplantation of autologous ocular surface epithelial stem cells via contact lens delivery: a prospective case series

IntroductionDepletion of limbal stem cells leads to a debilitating condition known as limbal stem cell deficiency, characterised by impaired corneal wound healing and poor vision. The aim of this study was to determine whether delivering progenitor cells on a contact lens is a viable and effective alternative to current transplantation techniques, which are complicated by biological and xenogeneic materials.MethodsSixteen eyes of 16 patients who had total (n = 14) and partial (n = 2) limbal stem cell deficiency (chemical burns, five eyes; iatrogenic causes, four eyes; aniridia, three eyes; trachoma-induced, two eyes; contact lens over-wear, one eye; and cicatrising conjunctivitis, one eye) and who had failed prior therapy were recruited prospectively into the study. Autologous limbal (n = 7) or conjunctival epithelial (n = 9) biopsies were harvested from patients and placed on the concave surface of silicone hydrogel contact lenses. Cells were expanded in culture with autologous serum and transplanted onto the ocular surface.ResultsRestoration of a transparent avascular and clinically stable corneal epithelium was attained in 10 of 16 eyes (63%) at a median follow-up time of 2.5 years (range of 0.8 to 5.8 years). Although minor complications occurred in two eyes of two patients because of contact lens insertion or removal, these were not associated with long-term sequelae.ConclusionsThis is the first and largest study to evaluate the mid-term outcomes of autologous limbal/conjunctival stem cell transplantation via a US Food and Drug Administration-approved contact lens, demonstrating that delivery of ocular progenitor cells via this procedure offers a viable, effective, and xeno-free alternative to current transplantation methodologies.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN012607000211460. Registered 17 April 2007.

Corneal blindness: a global problem.

Corneal blindness is an important, yet underreported cause of avoidable visual impairment worldwide, especially in developing countries. The World Health Organisation (WHO) estimates that corneal opacities (including trachoma) accounted for 7% of the world’s blind population in 2010, making it the 3rd most common cause of blindness. Although cataracts and glaucoma (in the elderly) are more common causes of visual impairment, corneal blindness affects all age groups and is a leading cause of irreversible visual impairment. An eye blind from scarring and vascularisation of the cornea, usually remains blind for life. In this issue of Clinical and Experimental Ophthalmology, Wang et al. present findings on corneal blindness from a large, population-based study of visual impairment in rural Heilong-Jiang province, China. Although there are numerous population-based studies detailing the major causes of blindness and low vision in both the developed and developing world, few published reports have thus far attempted to detail the different causes of corneal blindness. Rapoza et al. reported corneal infections (including trachoma) to be the leading cause of unilateral and bilateral corneal blindness in Central Tanzania, followed by vitamin A deficiency and measles. Unilateral corneal opacification had similar causes, with the important addition of trauma. Bowman et al. replicated these results in a population-based study in Gambia. Wang et al. here present findings on a large sample of 10 384 participants, representative of the rural Northern Chinese population, with a high overall response rate of 88.1%. Blindness was defined according to WHO criteria as a visual acuity of less than 3/60. All respondents underwent a screening examination, including measurement of best-corrected visual acuity (BCVA). Those achieving BCVA < 6/18 were subsequently referred for a more detailed examination. Although anterior segment examination was performed with a slit-lamp biomicroscope, fundus examination was carried with a hand-held ophthalmoscope only, without pupillary dilatation. This may have resulted in misclassification of the cause of blindness in a proportion of patients. Despite this shortcoming, the study presents some important findings. First, the majority (40%) of corneal blindness in this sample was acquired in childhood. Second, trauma (an entirely avoidable) cause of corneal blindness accounted for a third of all cases. Corneal opacification is the 3rd commonest cause of childhood blindness worldwide, after non-corneal causes such as congenital cataract and glaucoma. Unlike trachomatous corneal opacification, which results from repeated episodes of trachoma infection, corneal blindness in childhood is often due to a single episode of infection, such as ophthalmia neonatarum resulting from Neisseria gonorrhoea and Chlamydia trachomatis infections acquired from the mother’s genital tract at birth. During infancy and childhood, measles is another important cause of corneal blindness in the developing world, the impact of which is mediated through multiple mechanisms, including induction of acute vitamin A deficiency, measles keratitis, secondary bacterial or herpetic keratitis as well as the use of harmful traditional medicines. The WHO has ranked trachoma, corneal opacities, as well as childhood blindness, as priority eye diseases. Blind children have a lifetime of increased morbidity ahead of them. In addition, that lifetime can be very short, with up to 60% of blind children dying within 1 year of becoming blind. In this issue, Wang et al. report trauma as the second most common cause of corneal blindness in their population. In fact, corneal ulceration in developing countries is now considered a ‘silent epidemic’. Superficial corneal injuries from agriculture or domestic incidents led to blinding corneal ulceration due to delayed presentation and treatment. Indeed, in the developing world the majority of corneal ulcerations are the result of minor trauma and foreign bodies. This highlights the importance of public health education programs, targeting highrisk populations such as males, farmers and people with lower education. These programmes need to emphasize the importance of workplace safety, and timely hospitalization for corneal ulceration. In a previous study, also from China, Zhang and Wu demonstrated a lack of knowledge and awareness

The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities

Aims To investigate the effects of conditioned media from mesenchymal stem cells (MSC) on the wound healing activities of corneal stromal fibroblasts. Methods Cell cycle analysis and early stage activation of apoptosis, chemotactic chambers and fibroblast-populated type I collagen gels were used to assess corneal stromal fibroblast proliferation, migration and contraction, respectively. Fibroblasts were obtained from human donor corneas and MSC from fresh rat bone marrow. MSC conditioned media and fibroblast culture medium (FCM), with and without calf serum supplementation, were compared. Results MSC conditioned media and serum-free FCM had an inhibitory effect on the progression of corneal fibroblasts through the cell cycle. There was a significant increase in the number of cells in the G0–G1 phase for MSC conditioned media and serum-free FCM (p=0.001, p=0.97 respectively). Fibroblast migration and relaxed and stressed gel contraction were significantly inhibited by MSC conditioned media and serum-free FCM compared with FCM with serum (all p=0.001). Glucose and lactate analysis confirmed that these factors were not contributing to this effect. Conclusion MSC conditioned media was found to inhibit the wound healing activities of corneal stromal fibroblasts in vitro. Putative factors secreted by MSC could be developed for therapeutic use in corneal repair.

Chitosan-vancomysin composite biomaterial as a laser activated surgical adhesive with regional antimicrobial activity.

We have used laser irradiation to enhance the natural adhesiveness of chitosan to form a thin film surgical adhesive. Prevention of infection at surgical sites often utilizes systemic provision of antibiotics with reduced local efficacy and potential side effects. In the work reported here, we investigate the bactericidal properties of laser-irradiated chitosan films and their impregnation with the antibiotic vancomycin. Despite strong efficacy in solution, chitosan films showed no antimicrobial activity against representatives of common pathogens Escherichia coli , Staphylococcus aureus , and S. epidermidis . In contrast, a composite of chitosan adhesive and the antibiotic vancomycin showed therapeutically significant release profiles greater that the Minimum Bactericidal Concentrations (MBCs) for the Staphylococci over a 28 day period. These composite films had greater crystallinity, up to 28 ± 3 compared to 8.9 ± 2%, for its unblended counterpart. Despite a significant increase in material strength from 31.4 ± 4 to 77.5 ± 5 MPa, flexibility was still maintained with an elongation to break around 5 ± 2% and fold endurance of approximately 30 ± 3-folds. Laser irradiation had no apparent effect on the release or activity of the antibiotic which survived transient temperatures at the film-tissue interface during infrared irradiation of around 54 °C. Furthermore, significant adhesive strength was still apparent, 15.6 ± 2 KPa. Thus, we have developed a laser-activated bioadhesive with the potential to close wounds while facilitating the prevention of microbial infection through local release of antibiotic targeted to the site of potential infection.