Chondrocyte Death Quantified Using FDA-approved Fluorescein Is Increased Near Matrix Cracks Resulting From Traumatic Injury.

Kira D. Novakofski Rebecca Williams Lawrence Bonassar Lisa A. Fortier.
Cornell University Ithaca NY USA.

 K.D. Novakofski: None. R. Williams: None. L. Bonassar: None. L.A. Fortier: 3B; Consultant for Arthrex.

Introduction: Traumatic compression of cartilage results in chondrocyte death. Methods for quantifying cell death typically use reagents that are unsuitable for clinical use. Sodium fluorescein is an FDA-approved dye that is used topically and intravenously. Sodium fluorescein binds non-specifically to proteins1 and has shown potential in preliminary studies to label chondrocytes with compromised membranes. Validation of sodium fluorescein as a marker of dead chondrocytes would allow future clinical microscopy techniques to evaluate cartilage.
Chondrocyte death has been shown to occur near cracks of 50 µm in width at 18 hours after compressive injury. 2 Observations made at this time point might result from both mechanical effects and apoptosis. However cell death may occur immediately after injury prior to apoptosis which would affect future therapy development originally targeted toward apoptotic pathways. Therefore the purpose of this study was to determine if sodium fluorescein could be used as an indicator of cell death and to determine if cell death was increased near cracks immediately after injury.
Methods: Tissue collection and traumatic injury. Osteochondral blocks (OCBs) from the distal 3rd metacarpus were collected normal horses (n=5) aged 4-6 years and incubated in phenol red free MEM. OCBs were briefly removed from the media and mounted between a custom-designed specimen-chamber and 2.25 mm diameter impactor on an EnduraTEC ELF3200 test frame (EnduraTec Minnetonka MN). The medial condyle was subjected to a single compression of 30 MPa within 1 sec. OCBs were then incubated in MEM for 1 h at 37°C.
Multiphoton imaging and fluorescein validation. OCBs were placed in 1 µM fluorescein in PBS for 10-15 min and then imaged within the solution. Images of fluorescein staining were acquired of the cartilage in cross section by exciting at 780 nm with a Ti:sapphire laser (Newport Irvine CA); emission signal was collected using a 380-540 nm filter. To validate fluorescein as a dead cell stain 1 µM ethidium homodimer-1 (EthD-1) was added and incubated for 30 min. Emission signal was acquired again with a 560-650 nm filter. Fluorescein was validated as an indicator of cell death and staining with EthD-1 was discontinued (n=9 sites n=1 OCB). The remainder of the data was collected using only fluorescein.
Image analysis. Images of regions with cracks were acquired with 380-500 and 500-550 nm emission filters 10 µm below the articular surface in the transverse plane. Each of the first three cells that were found within 100 µm distance from each crack was counted around the crack perimeter (n=13 sites n=5 OCBs). Cell death in injured sites was compared to cell death in uninjured lateral condyle controls (n=13 sites n=5 OCBs). Cracks were not observed in control sites.
Results: Serial staining with sodium fluorescein and EthD-1 verified the use of fluorescein as a dead cell indicator. Fluorescein correlated with EthD-1 staining with slope=1.049 and an r2=0.929 (Figure 1).
Cell death within 100 µm of cracks was significantly increased(95% CI 64.0-83.4% p<0.0001 Figure 2) with 73.7% of cells being dead compared to corresponding control sites with cell death of 1.0% (95% CI 0-2.2%).
Discussion: Sodium fluorescein was found to strongly correlate with EthD-1 staining. This result suggests the potential use of sodium fluorescein in the clinic as a diagnostic tool to identify cell death. Cell death can occur in isolated locations and was found to be increased immediately in the regions near cracks that were as small as 2 µm. Previous work has shown increased cell death near large cracks at 18 hours after injury; 2 however the present study shows that cell death occurs near small cracks and at an early time point within 1 hour of injury. This death likely results from cellular or matrix disruption whereas at a later time point when apoptosis likely contributes.
Our findings suggest that quantifying local cell death with an FDA-approved dye is possible and should help with the early diagnosis of cartilage disease.
Significance: Identifying dead chondrocytes in cartilage in the clinical setting is challenging but new imaging methods are being developed using fluorescent labeling that is safe for patient use. Sodium fluorescein is FDA-approved and can quantify dead chondrocytes including increased cell death near small cracks which may affect diagnostic and therapeutic management.
Acknowledgments: The authors would like to thank Robert Bowles for his assistance in developing the injury model. The study was supported by the Harry M. Zweig Foundation for Equine Research (LAF) and grant TL1RR000459 of the Clinical and Translational Science Center at Weill Cornell Medical College (KDN).
References: 1. Andersson LO Rehnström A Eaker DL. Studies on “Nonspecific” binding. European Journal of Biochemistry. 1971;20(3):371-380. 2. Lewis JL Deloria LB Oyen‐Tiesma M Thompson Jr RC Ericson M Oegema Jr TR. Cell death after cartilage impact occurs around matrix cracks. Journal of Orthopaedic Research. 2003;21(5):881-887."