Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs.

Cell-Free Biological Approach for Corneal Stromal Wound Healing

Corneal opacification is the fourth most common cause of blindness globally behind cataracts, glaucoma, and age-related macular degeneration. The standard treatment of serious corneal scarring is corneal transplantation. Though it is effective for restoring vision, the treatment outcome is not optimal, due to limitations such as long-term graft survival, lifelong use of immunosuppressants, and a loss of corneal strength. Regulation of corneal stromal wound healing, along with inhibition or downregulation of corneal scarring is a promising approach to prevent corneal opacification. Pharmacological approaches have been suggested, however these are fraught with side effects. Tissue healing is an intricate process that involves cell death, proliferation, differentiation, and remodeling of the extracellular matrix. Current research on stromal wound healing is focused on corneal characteristics such as the immune response, angiogenesis, and cell signaling. Indeed, promising new technologies with the potential to modulate wound healing are under development. In this review, we provide an overview of cell-free strategies and some approaches under development that have the potential to control stromal fibrosis and scarring, especially in the context of early intervention.

One-year Observational Study of an Iris Camera Corneal Opacification Measurement and Pentacam Densitometry in Keratoconus after Corneal Crosslinking

Purpose: To investigate levels of corneal haze in adult patients with keratoconus treated by corneal crosslinking, using Iris camera and Corneal densitometry imaging. Methods: This was a prospective, observational cross-sectional study on patients attending the Manchester Royal Eye Hospital outpatient department. Iris camera, Pentacam densitometry and visual acuity measurements were collected from patients in two groups. In group A, patients were assessed before and after crosslinking (CXL), whereas in group B, measurements were made for an extended period after their CXL had been performed. Results: Group A consisted of 13 eyes of 13 patients, and group B consisted of 41 eyes of 41 patients. In group A average age was 26.69 (7.19) years. Average time of measurements taken before CXL was 1.85 (0.80) months and after CXL 12.69 (0.63) months. When comparing values before CXL and after, there was a significantly decreased COM score (P=0.001) and Pentacam Corneal densitometry measurement (P=0.003) representing improved corneal clarity. There was a corresponding significant improvement in vision in all eyes in group A after CXL procedure, and 100% of eyes reached a BCVA of ≤20/50 (≤0.4 LogMAR) (P=0.002). In group B, the mean first examination time was post-treatment 3.24 (1.48) months and for the second examination, post-treatment 12.56 (0.81) months. Between these two visits, there was a significant decrease in COM score (P=0.002) and corneal densitometry (P=0.0004) as well as improvement in BCVA (P=0.0002). In this group also, 100% of eyes reached a BCVA of ≤20/50 (≤0.4 LogMAR) by the time of their last follow up. Conclusion: There is a significant improvement in corneal opacification measures using both Iris camera and densitometry when assessing before and after CXL and also sequentially 3 and 12 months after the procedure. The iris camera represents a promising, portable and practical means of assessing and monitoring corneal clarity for this purpose.

Evolution of corneal flattening after repeated corneal cross-linking during a 6-year follow-up

Purpose: To report the evolution of corneal flattening after repeated corneal cross-linking (CXL) in a patient with progressive keratoconus during a 6-year follow-up. Methods: Case report. Results: A 27-year-old female underwent CXL for progressive keratoconus. Postoperatively, corneal topography revealed keratoconus progression with an increase of 1.20 diopters (D) in maximum keratometry (Kmax) and CXL was repeated. After the second treatment, a continuing significant corneal flattening (up to 16.00 D in Kmax) was observed during the first 5 years followed by stabilization during the last sixth year of follow-up. Both uncorrected and corrected distance visual acuity were improved while corneal thickness was decreased. There were no complications such as corneal opacification or endothelial cells decrease during the follow-up period. Conclusion: Repeated CXL can induce an excessive corneal flattening more pronounced during the first years of follow-up followed by stabilization thereafter.

Corneal Opacification and Spontaneous Recovery following Injection of Healon5 into the Corneal Stroma during Intervention for Postoperative Hypotony

We report the self-limited nature of corneal opacification after accidental injection of Healon5 into the corneal stroma. A 52-year-old male with a new diagnosis of severe stage, primary open-angle glaucoma underwent successful trabeculectomy OS, which was complicated by ocular hypotony and shallow anterior chamber (AC) on postoperative day 1. Healon5, a hyaluronic acid-containing viscoelastic device, was accidentally introduced into the corneal stroma during attempted injection into the AC. The cornea hydrodissected and opacified, leading to precipitous loss of best-corrected visual acuity (BCVA). The corneal opacification spontaneously resolved over a 7-month period without specific intervention. During this period, the patient also underwent cataract extraction with posterior chamber intraocular lens placement and YAG capsulotomy, after which his BCVA returned to approximately baseline. Though intrastromal injection of sodium hyaluronate-containing material has been reported elsewhere, this complication with Healon5 use specifically has yet to be described in the literature and may occur in any procedure involving Healon5 in the AC. This case report is important, since the precipitous loss of BCVA can be alarming to the ophthalmologist and the patient. The affected patient may be counseled that the opacification should improve with time.

Indications and prognosis for keratoplasty in eyes with severe visual impairment and blindness due to corneal disease in India

PurposeTo describe the indications and prognosis for keratoplasty in eyes with severe visual impairment and blindness due to corneal diseases in India.MethodsThis hospital-based cross-sectional study included 1 057 215 new patients presenting to a multitier ophthalmology network from 2016 to 2018. All patients with a central corneal pathology and best corrected visual acuity of less than 20/200 in the affected eye(s) were identified as cases and grouped according to laterality. The main outcome measure was the prognosis for keratoplasty, based on the surgical indication.ResultsA total of 28 824 (2.73%) patients were identified with severe visual impairment or blindness due to corneal diseases; of which, 22 582 (78.3%) had unilateral and 6242 (21.7%) had bilateral affliction. Overall, 85% of the corneal pathologies were due to avoidable causes, which accounted for 97% and 63% of the unilateral and bilateral cases, respectively (p<0.0001). The most common aetiologies were microbial keratitis (27.2%) and corneal opacification (25.5%) in unilateral cases; and corneal opacification (38.2%) and ectasias (14.5%) in bilateral cases. Overall, 60.1% of affected eyes carried a fair to poor prognosis for keratoplasty. However, while in unilateral cases only 29.9% of eyes carried good to excellent prognosis for keratoplasty, in bilateral cases, 58% of eyes carried good to excellent prognosis for keratoplasty (p<0.0001).ConclusionsThe indications and prognosis for keratoplasty varied greatly based on whether the affliction was unilateral or bilateral. Most of the eyes affected by corneal visual impairment or blindness, however, carried an unfavourable prognosis for keratoplasty.

PECULIARITIES OF BACTERIAL KERATITIS IN PATIENTS WITH DIABETES MELLITUS DEPENDING ON THE SEVERETY OF DIABETIC POLINEUROPATHY

Summery. The dependence of the peculiarities of bacterial keratitis on the severity of DPN was proven with the help of 15 patients with type 1 diabetes mellitus. During all periods of observation, the severity of conjunctival hyperemia, the size of erosion and the threshold of corneal sensitivity at the stage of severe complications exceeded the corresponding indicators of patients with asymptomatic DPN. Normalization of conjunctival color in all patients with severe complications occurred at 8 days, and reepithelialization of the cornea 4 days later than in asymptomatic DPN. On the last 22nd day of observation, the corneal opacification of the peripheral localization remained in 33.3% of patients (2 patients) with the stage of severe complications, whereas in all patients with asymptomatic DPN the cornea was already transparent on the 14th day of observation. The threshold of corneal sensitivity at 22nd day in patients with severe complications was 35.3% higher than with asymptomatic DPN (p <0.05). So, patients with severe complications DPN with bacterial keratitis show a more pronounced violation of corneal innervation, which manifests by decrease of its sensitivity, and leads to prolongation of the duration of the disease.