title: Identification of cartilage injury using quantitative multiphoton microscopy.
abstract: OBJECTIVE
  Cartilage injury can lead to post-traumatic osteoarthritis (PTOA). Immediate post-trauma cellular and structural changes are not widely understood. Furthermore, current cellular-resolution cartilage imaging techniques require sectioning of cartilage and/or use of dyes not suitable for patient imaging. In this study, we used multiphoton microscopy (MPM) data with FDA-approved sodium fluorescein to identify and evaluate the pattern of chondrocyte death after traumatic injury.
  
  
  METHODS
  Mature equine distal metacarpal or metatarsal osteochondral blocks (OCBs) were injured by 30 MPa compressive loading delivered over 1 s. Injured and control sites were imaged unfixed and in situ 1 h post-injury with sodium fluorescein using rasterized z-scanning. MPM data was quantified in MATLAB, reconstructed in 3-D, and projected in 2-D to determine the damage pattern.
  
  
  RESULTS
  MPM images (600 per sample) were reconstructed and analyzed for cell death. The overall distribution of cell death appeared to cluster into circular (n = 7) or elliptical (n = 4) patterns (p = 0.006). Dead cells were prevalent near cracks in the matrix, with only 26.3% (SE = 5.0%, p < 0.0001) of chondrocytes near cracks being viable.
  
  
  CONCLUSIONS
  This study demonstrates the first application of MPM for evaluating cellular-scale cartilage injury in situ in live tissue, with clinical potential for detecting early cartilage damage. With this technique, we were able to uniquely observe two death patterns resulting from the same compressive loading, which may be related to local variability in matrix structure. These results also demonstrate proof-of-concept MPM diagnostic use in detecting subtle and early cartilage damage not detectable in any other way.
authors.affiliation: Department of Clinical Sciences, Cornell University, Ithaca, NY, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: Novakofski
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Williams
authors.affiliation: Department of Clinical Sciences, Cornell University, Ithaca, NY, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Fortier
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853, USA.
authors.forename: H O
authors.initials: HO
authors.lastname: Mohammed
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: W R
authors.initials: WR
authors.lastname: Zipfel
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA. Electronic address: lb244@cornell.edu.
authors.forename: L J
authors.initials: LJ
authors.lastname: Bonassar
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000293:injuries* / Q000473:pathology; D016923:Cell Death / Q000502:physiology; D019902:Chondrocytes / Q000473:pathology; D004195:Disease Models, Animal; D042241:Early Diagnosis; D005240:Feasibility Studies; D006736:Horses; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008663:Metacarpus / Q000293:injuries / Q000473:pathology; D008682:Metatarsal Bones / Q000293:injuries / Q000473:pathology; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D013314:Stress, Mechanical; D016474:Weight-Bearing
keywords: Cartilage; Imaging; Injury; Multiphoton microscopy; Post-traumatic osteoarthritis