Surface quality assessment of explanted keratoprostheses using confocal and scanning electron microscopy

Abstract

 

Purpose

 

To evaluate the effects of the irregular surfaces of Boston Type I keratoprostheses after explanation using confocal and scanning electron microscopy.

 

Methods

 

Failed Boston Type I Keratoprostheses (KPro) were collected from patients undergoing KPro explantation or exchange at Bascom Palmer Eye Institute, Miami, FL, USA. In the operating room, the KPro samples were placed in a container with balanced salt solution immediately after removal. Fluorescent confocal microscopy was performed on the fresh, un-fixed KPro samples to visualize the microbial adherence and cellular growth. A live/dead green/red fluorescent stain was used along with a Leica 5PS confocal microscope. Images were taken across the entire anterior and posterior surfaces of the KPro samples to characterize the complete KPro surface. The optical surfaces of the KPro were imaged with bright field illumination of the confocal microscopy. After confocal microscopy, the KPro sample is fixed in 10% formalin, immersed in PBS buffer, dehydrated in a graded series of ethanol, dried in HMDS, and sputter-coated with Palladium for scanning electron microscopy (SEM). Images of the anterior and posterior surfaces of the KPro were obtained using SEM at multiple magnifications (30x-5000x).

 

Results

 

Confocal microscopy and SEM images showed rough surfaces on all regions of the keratoprostheses. The confocal microcopy revealed cellular growth in areas of more irregularities. The high magnification SEM images showed many bacteria and biofilm colonies attached to the KPros. In one case, the patient also had an intraocular lens (IOL) which was analyzed as was the KPro to relate surface features to microbial adherence. The IOL had super polished surfaces with almost no microbial adherence.

 

Conclusions

 

Dual imaging approaches in this ongoing study enabled an accurate evaluation of the failed keratoprostheses, and thus better elucidated the mechanisms that lead to their explantation.  

 

Confocal microscopy images of external and internal KPro surfaces shows cellular activity and biofilm

 

 

SEM images of external and internal KPro surfaces. The retroprosthetic membrane traverses through the holes extends to the anterior surface of the backplate.