PATCH GRAFTING ADULT HUMAN BRUCH'S MEMBRANE EXPLANTS TO REPAIR FOCAL DEFECTS IN THE HOST BRUCH'S MEMBRANE (BM): AN INITIAL STEP OF TISSUE ENGINEERING FOR AGE-RELATED MACULAR DEGENERATION

Abstract

Purpose : To test the efficacy of a non-degradable biocompatible photopolymerizable hydrogel (BPG) in patch-grafting adult human BM explants onto focal defects of host BM ex vivo.

Methods : Freshly enucleated (≤ 6hrs) porcine eyes were obtained from a local slaughterhouse. Anterior segment and sensory retina were dissected out and RPE cells were removed by 0.02 N ammonium hydroxide incubation for 20 minutes. Exposed BM was mechanically scratched to mimic uneven structural damage created after foveal membranectomy. BM defects were then patched with 7.75 mm circular grafts of adult human BM prepared from freshly (<24 hours) harvested adult human eyes. 5-10 μl of a non-degradable BPG composed of 100 mg/mL poly(ethylene glycol) diacrylate (PEG-DA) with 10 mg/mL hyaluronic acid (HA, viscosity 5200 mP.sec) was used to affix BM grafts onto host BM. The strength of the attachment was tested by applying traction on human BM patch graft through a 100 μm thick mixed cellulose ester membrane. Gradually increasing force was applied through a pulley system to determine the breaking point of the attachment between the graft and the host BM. Another set of eyecups were incubated at 37^○C for up to 17 days. Eyecups were examined daily for spontaneous separation of the patch graft. The strength of the BM graft to vertical traction was also calculated at the end of the observation period.

Results : Biogel solidified ≤10 minutes of exposure to a 10 lumens of green LED light. None of the grafts spontaneously detached. Patched grafts resisted vertical stresses greater than 806.7 ± 467.5 Pa. Patched BM explants remained adherent onto host BM throughout the observation period of 17 days without a change in the adherence force. Photomelding the patch BM grafts onto host BM did not adversely affected the viability of the grafted iPS-RPE cells ex vivo.

Conclusions : Patch grafting of human BM onto host BM with a non-degradable BPG can seal and repair focal BM defects in patients with age-related macular degeneration. BM grafts can act as a substrate to culture and transplant retinal cells into the host subretinal space. Patch grafts of BM can provide vertical stability and create a natural substrate for proper differentiation and functioning of the transplanted cells.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

A circular patch of human Bruch's membrane grafted onto pig Bruch's membrane