Disclosures: C.B. Raub: None. B.C. Hansen: None. T. Yamaguchi: None. M.M. Temple-Wong: None. K. Masuda: None. R.L. Sah: None.

Transcription

1 En Face Microscopy of Rabbit Knee Articular Cartilage Following Anterior Cruciate Ligament Transection Reveals Early Matrix Damage, Chondrocyte Loss and Cloning Christopher B. Raub, PhD, Bradley C. Hansen, MS, Tomonori Yamaguchi, MS, Michele M. Temple-Wong, PhD, Koichi Masuda, MD, Robert L. Sah, MD, PhD. University of California, San Diego, La Jolla, CA, USA. Disclosures: C.B. Raub: None. B.C. Hansen: None. T. Yamaguchi: None. M.M. Temple-Wong: None. K. Masuda: None. R.L. Sah: None. Introduction: In the rabbit ACL transection (ACLT) model of osteoarthritis, cartilage erosion in both the medial and lateral femoral condyles (MFC, LFC) occurs by 8 weeks 1, but the structural nature of the cartilage damage at earlier times is unclear. In articular cartilage with minimal and overt fibrillation, linear clefts and irregularly-shaped depressions are evident by light 2, 3 and electron microscopy 4, 5. In articular cartilage from joints with osteoarthritis (OA), cellularity is less than normal 6, 7, and superficial chondrocytes are present as brood clones 7, 8. It may be that joints that have been injured and are predisposed to OA exhibit microscopic alterations of the articular surface and superficial chondrocytes that are similar to, or distinct from, alterations associated with aging and aging-associated OA. The aims of this study were to characterize and distinguish between the microscopic patterns, and the spatial association between surface damage and altered chondrocytes, in non-operated and ACLT rabbit knees at an early time-point following surgery, before the appearance of macroscopic fibrillation. Methods: In an IACUC-approved study, New Zealand White rabbits were subjected to unilateral ACLT, and at 4 weeks posttransection (14 ± 4 months old, 4.4 ± 0.5 kg, mean ± SD, n=6 animals) the femoral condyles of ACLT and contralateral nonoperated knees were collected and analyzed. Microscopic patterns of India ink-marked surfaces and superficial zone chondrocyte nuclei just beneath the articular surface were assessed by both en face microscopy (India ink and chondrocyte nucleus stain) and traditional sagittal histology (safranin-o and H&E stains). En face microscopy images were processed using Matlab to create masks from which quantitative geometrical parameters were calculated, and semiquantitative scores defined to assess matrix damage and altered cell fate, with a higher score indicating more alteration from normal. Also, reflected light and fluorescence microscopy images were co-registered, and chondrocyte density, clustering and orientation adjacent to India ink features were assessed quantitatively and by visual inspection of co-registered en face and sagittal section images. Twofactor repeated measures ANOVA (or Kruskal-Wallis tests) were performed to compare non-operated vs. ACLT-treated knees, and anterior vs. posterior condyle sites. If two-factor tests were significant (p<0.05), Wilcoxon signed rank t-tests were used for post-hoc testing. Correlation analyses were performed between both India ink and cell parameters and sub-scores, with Spearman s correlation coefficient (ρ) calculated and tested for significance. Results: Relative to non-operated knees, in en face microscopy, ACLT knees exhibited more extensive and connected surface damage patterns, chondrocyte loss, and clusters (>9) of cells (Figure 1). Specifically, the area fraction of India ink features was greater for ACLT than non-operated knees in the anterior MFC (6.99 ± 2.11% vs. 2.6 ± 2.0%) and posterior MFC (15.8 ± 4.8% vs. 1.9 ± 0.9%). The apparent cell density was lower in the ACLT than non-operated posterior MFC (510 ± 220/mm 2 vs. 700 ± 110/mm 2 ) and posterior LFC (390 ± 170/mm 2 vs. 620 ± 100/mm 2 ). Traditional histological sections indicated only mild disruption at the articular surface (Figure 2). Surface scores were correlated between posterior and anterior sites (ρ=0.57), LFC and MFC (ρ=0.54), and sub-score metrics of cellularity and matrix damage (ρ=0.65). Chondrocyte clusters aligned parallel to the orientation of adjacent India ink features (Figure 3). Discussion: Although non-operated condyles contained some surface damage revealed by India ink, the patterns of damage in condyles of ACLT-treated knees were more extensive and distinct, in that the surface features were often connected and associated with local (within 50 μm) cell loss and cloning, and oriented similarly to cell nucleus axes. These observations suggest extension and joining of fissures along planes between collagen fibril bundles, similar to patterns of split lines 9, or lattice-like patterns in minimally-fibrillated cartilage 2. The greater extent of surface damage and associated chondrocyte loss and cloning in ACLT-treated knees support a role for biomechanical modification of superficial cartilage microstructure and chondrocytes, which together with the altered regulatory and enzymatic environment of the synovial joint after injury, ultimately damage the cartilage collagen network. Significance: Early deterioration of articular cartilage following ACLT in rabbits involves alterations to the surface matrix and superficial chondrocytes, detected sensitively by en face light microscopy. Targeted detection of these alterations may be useful both in translational studies of OA as well as in arthroscopic diagnosis and monitoring of cartilage deterioration. Acknowledgments: We thank Iliya Goldberg and David Berry for technical assistance. References: 1. Yoshioka+. Osteoarthritis Cartilage. 4: 87-98, 1996.

2 2. Meachim. Ann Rheum Dis. 31: , Bae+. Osteoarthritis Cartilage. 16: 841-5, Clark. J Anat. 171: , Clarke. J Bone Joint Surg Br. 53: , Temple+. Osteoarthritis Cartilage. 15: , Rolauffs+. J Struct Biol. 162: , Schumacher+. Anat Rec. 266: 241-8, Meachim+. J Anat. 118: , 1974.

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4 ORS 2014 Annual Meeting Poster No: 0356

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