title: Femtosecond laser cutting of endothelial grafts: comparison of endothelial and epithelial applanation.
abstract: OBJECTIVE
  Stromal surface quality of endothelial lamellae cut for endothelial keratoplasty with a femtosecond laser (FSL) with epithelial applanation remains disappointing. Applanation of the endothelial side of the cornea, mounted inverted on an artificial chamber, has therefore been proposed to improve cut quality. We compared lamellar quality after FSL cutting using epithelial versus endothelial applanation.
  
  
  METHODS
  Lamellae were cut with an FSL from organ-cultured corneas. After randomization, 7 were cut with epithelial applanation and 7 with endothelial applanation. Lamellae of 50-, 75-, and 100-μm thickness were targeted. Thickness was measured by optical coherence tomography before and immediately after cutting. Viable endothelial cell density was quantified immediately after cutting using triple labeling with Hoechst/ethidium/calcein-AM coupled with image analysis with ImageJ. The stromal surface was evaluated by 9 masked observers using semiquantitative scoring of scanning electronic microscopy images. Histology of 2 samples was also analyzed before lamellar detachment.
  
  
  RESULTS
  Precision (difference in target/actual thickness) and thickness regularity [coefficient of variation (CV) of 10 measurements] were significantly better with endothelial applanation (precision: 18 μm; range, 10-30; CV: 11%; range, 8-12) than with epithelial applanation (precision: 84 μm; range, 54-107; P = 0.002; CV: 24%; range, 13-47; P = 0.001). Endothelial applanation provided thinner lamellae. However, viable endothelial cell density was significantly lower after endothelial applanation (1183 cells/mm2; range, 787-1725 versus 1688 cells/mm2; range, 1288-2025; P = 0.018).
  
  
  CONCLUSIONS
  FSL cutting of endothelial lamellae using endothelial applanation provides thinner more regular grafts with more predictable thickness than with conventional epithelial applanation but strongly reduces the pool of viable endothelial cells.
authors.affiliation: *Corneal Graft Biology, Engineering and Imaging laboratory, EA2521, SFR143, Faculty of Medicine, University Jean Monnet, Saint-Etienne, France; †Department of Ophthalmology, University Hospital, Besançon, France; ‡Department of Ophthalmology, University Hospital, Saint-Etienne, France; §Manutech Ultrafast Surfacing Design, Pole Optique Rhône-Alpes, Saint-Etienne, France; ¶Department of Pathology, University Hospital, Saint-Etienne, France; and ‖Institut Universitaire de France, Boulevard Saint-Michel, Paris, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Bernard
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: He
authors.forename: Anne Sophie
authors.initials: AS
authors.lastname: Gauthier
authors.forename: Marie Caroline
authors.initials: MC
authors.lastname: Trone
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Baubeau
authors.forename: Fabien
authors.initials: F
authors.lastname: Forest
authors.forename: Jean Marc
authors.initials: JM
authors.lastname: Dumollard
authors.forename: Michel
authors.initials: M
authors.lastname: Peocʼh
authors.forename: Gilles
authors.initials: G
authors.lastname: Thuret
authors.forename: Philippe
authors.initials: P
authors.lastname: Gain
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002452:Cell Count; D002470:Cell Survival; D057111:Descemet Stripping Endothelial Keratoplasty; D004728:Endothelium, Corneal / Q000473:pathology / Q000601:surgery*; D019573:Epithelium, Corneal / Q000473:pathology / Q000601:surgery*; D006801:Humans; D053685:Laser Therapy; D054018:Lasers, Excimer / Q000627:therapeutic use; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D014019:Tissue Donors; D020858:Tissue and Organ Harvesting / Q000379:methods*; D009927:Tissue and Organ Procurement; D041623:Tomography, Optical Coherence