Purpose: Human articular cartilage (hAC) exhibits degeneration that is site-specific and exhibits six characteristic patterns of early surface damage. These hAC patterns were distinguished by staining the cartilage surface with India Ink and photographing en face with film providing ∼(4 um)2 pixel resolution. However, the mechanisms for the initiation and progression of such damage are unclear. Modern 2-D digital imaging allows quantitative imaging over large areas, and 3D histology (3DH) allows structural visualization at ∼(0.5-1 μm)3 voxel resolution. Animal models of post-traumatic osteoarthritis (PTOA) may help elucidate the underlying mechanical and/or biological mechanisms of such damage. In the rabbit ACL transection (rACLT) model of PTOA, cartilage damage progresses steadily over 9 weeks. The aims of the present study were to analyze site-specific AC damage at four weeks after rACLT by (1) High Resolution Digital Imaging of India Ink Staining (designated here as HiReD-IIIS, “hired eyes”), (2) elucidate the 3D pattern of surface damage by 3DH, and (3) assess the extent of inhomogeneity and anisotropy of cartilage surface damage at weight-bearing sites. Methods: Rabbit Samples. With IACUC approval, ten adult female New Zealand White rabbits (11-19 mo, weight = 3.8-5.2 kg) were subjected to unilateral (right knee) ACLT to induce PTOA. At day 28, rabbits were sacrificed, the ACLT and contralateral non-operated left (CTRL) knees were harvested, and the FCs were exposed. High Resolution Digital Imaging. AC surfaces were aligned, dabbed with a 1:5 dilution of India Ink in phosphate buffered saline (PBS) + proteinase inhibitors, and digitally imaged at high resolution (4 μm)2/pixel. Custom Image Processing. Gray-scale images were normalized to 0 (darkest) and 1 (brightest) using standards, and cropped to isolate the weight bearing regions: posteromedial [PostMed] and posterolateral [PostLat] on each condyle. Surface damage area was calculated in two locations of the size ∼15mm2 on a) medial and b) lateral aspects of each weight bearing region (PostMed-M, PostMed-L, PostLat-M and PostLat-L) using 2-D morphometric analysis (adaptive thresholding by mean, despeckling, and morphological operations [closing]), normalized to the overall selected condyle area. The overall surface damage orientation, defined relative to horizontal axis ranging from [-90o, 90o], was approximated by vector analysis. Statistics. Damage area data are shown as mean ± SE. At each weight bearing region, ACLT and CTRL were compared for normalized damage area by two-way ANOVA, and for damage area and orientation association by Pearson correlation. Significance was taken as p<0.05. Results: HiReD-IIIS revealed site-specific articular cartilage (AC) deterioration patterns (Figure 1) including [1-TCL] transverse curvilinear lines (perpendicular to articulation axis), [2-PCL] parallel curvilinear lines (parallel to articulation axis), [3-RST] reticular sawtooth, and [4-R] ravine (tortuous/curvilinear contours), in the (L)ateral and (M)edial femoral condyle (FC) load-bearing regions, confirmed by 3DH. ACLT induced detectable damage, increasing normalized damage area (Figure 2A) by 4-30% in PostLat-M site, with 80% of the FCs’ lesion area concentrated between 5-25%, as compared to CTRL (p<0.0001). Surface damage patterns in all locations had an increasing trend in area with PostLat-M and PostMed-L reaching a maximum of 35% and 23%, respectively. Damage area was negatively correlated with the overall lesion orientation in the PostLat-L region (r = -0.66). Surface damage, R, was the primary pattern at PostLat-M, appearing at different states of progression. On PostLat-L, normalized damage areas of <5% and 5-18% corresponded with TCL and RST patterns, respectively (Figure 2B). On PostMed-L, lesion areas of <10% matched with PCL and TCL, and ≥10% with RST patterns. Conclusions: The integrated high-resolution image acquisition and morphometric lesion analysis revealed (1) a trajectory for evolution of TCL into RST in the PostLat-L and of TCL and PCL into RST on the PostMed-L regions, and (2) progressive occurrence of site-specific lesions that are more prominent on the PostMed-L and PostLat-M, which could be indicative of altered contact between joint constituents, leading to abnormal loading modes and directions in a destabilized joint.View Large Image Figure ViewerDownload Hi-res image Download (PPT)