Nanomechanical Symptoms in Cartilage Precede Histological Osteoarthritis Signs after the Destabilization of Medial Meniscus in Mice Basak Doyran 1, Wei Tong 2, Qing Li 1, Haoruo Jia 2, Xianrong Zhang 3, Motomi Enomoto-Iwamoto 2,4, Ling Qin 2, Lin Han 1. 1 School of Biomedical Engineering, Sciences and Health Systems, Drexel University, Philadelphia, PA, USA, 2 Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA, USA, 3 Wuhan University, Wuhan, China, 4 Department of Surgery, The Children s Hospital of Philadelphia, Philadelphia, PA, USA. Disclosures: B. Doyran: None. W. Tong: None. Q. Li: None. H. Jia: None. X. Zhang: None. M. Enomoto- Iwamoto: None. L. Qin: None. L. Han: None. Introduction: Murine model is widely used for investigating the pathogenesis and progression of knee osteoarthritis (OA) [1]. Current evaluation of murine OA relies on semi-quantitative signs measured by histological [2] or radiological [3] assays, including cartilage surface fibrillation, decreased proteoglycan staining, joint space thinning, etc. These signs are macro- or microscopic symptoms of synovial joint abnormality. While they clearly illustrate joint morphological changes during OA progression, they do not allow us to identify an initial alteration in prior to macroscale structural changes of articular cartilage, or, to measure cartilage mechanical properties that are directly relevant to its tissue function. In this study, to find a more sensitive and function-relevant indicator of OA, we quantified the effective indentation modulus of femoral condyle cartilage in a murine OA model involving destabilization of the medial meniscus (DMM). Methods: Surgeries were performed on skeletally mature 12-weeks old male C57BL/6J wild-type mice on the right hind knee for DMM and on the left hind knee for the Sham control, following the procedures described previously [4]. Briefly, immediately after anesthesia, in DMM, the joint capsule was opened and the medial meniscotibial ligament was cut to destabilize the meniscus without damaging other tissues. In Sham surgery, the joint capsule was opened in the same fashion but without any further damage. For nanoindentation, mice were euthanized 2-4 weeks after the surgery (n 5 at each time point), and medial condyles were harvested and stored in phosphate buffer saline (PBS, IS = 0.15 M, ph = 7.4) with protease inhibitors (Pierce) at 4ºC for < 24 h. Atomic Force Microscopy (AFM)- based nanoindentation was performed on the superficial zone of cartilage using a borosilicate colloidal spherical tip (R 5 μm, nominal spring constant k 7.4 N/m, AIO-TL tip C, NanoAndMore) and a Dimension Icon AFM (BrukerNano), following previously established procedures [5]. For each condyle cartilage, at least 10 different indentation locations were tested up to an indentation depth of 1 µm at 1 µm/s and 10 µm/s rates. Effective indentation modulus, E ind (MPa), was calculated from the loading portion of indentation force-depth curves using the Hertz model (Fig. 1a). In histology, mice were euthanized 2-12 weeks after the surgery. Whole joints of both knees (n 5) were harvested, fixed in 4% paraformaldehyde (Fisher), decalcified in 10% EDTA (Amresco) and embedded in paraffin. Serial 4 µm thick sagittal sections were cut across medial side of the joint and two sections within every consecutive six sections were stained with Safranin-O/Fast Green and hematoxylin. From each knee, approximately 15 sections were obtained and scored by at least two masked observers using the modified Mankin s
method [2]. Each section was assigned a score, which was the summation of individual structures, involving cartilage (0-5), chondrocytes (0-3), Safranin-O staining (0-5) and tidemark (0-1). Mann-Whitney U test was performed to study the differences in E ind and histological scores between results from DMM and Sham surgeries, where a p-value < 0.05 was considered statistically significant. Results: Using AFM-nanoindentation, we found significantly lower E ind for the right knee that underwent DMM, compared to the left knee that underwent the Sham, at 4 weeks after the surgery (e.g., Fig. 1b). Furthermore, cartilage exhibited significantly lower in E ind at both 2 and 4 weeks after DMM compared to the Sham control (n 5 for each time point) (Fig. 2). This DMM-induced weakening appeared to be consistent for all the mice tested in this study. In contrast, consistent with the literature [8], we did not find a significant increase in histology-based Mankin scores until 8-12 weeks after DMM (Fig. 3). The histology results after 2-4 weeks were not shown since no differences were found compared to the Sham control. Discussion: Decrease in E ind of cartilage superficial layer appeared as early as 2 weeks after DMM, preceding the macroscopic OA signs measured by histology (8-12 weeks). This observation suggests that cartilage nanomechanical properties have the potential to be used as an early indicator of OA. This method is advantageous over conventional assays as it focuses on changes at smaller length scales and the modulus is a direct measure of cartilage biomechanical function. The superficial layer modulus, E ind, is governed by both the concentration and hierarchical structure of major matrix constituents, i.e., transversely aligned type II/IX/XI collagen fibrillar mesh, and proteoglycans including aggrecan and lubricin [6]. It is likely that the mechanical instability introduced by DMM disrupts superficial layer nanostructure before leading to inflammatory chondrocyte responses and later on macroscopic OA symptoms. As a result, the mechanical weakening could be a direct manifestation of the aberrant nanostructure. The early decrease in E ind is also consistent with our recent study on the phenotype of chondroadherin-null murine cartilage [5]. In this model, cartilage superficial layer exhibits significantly lower E ind than wild-type despite the absence of histological differences. This early nanomechanical symptom is also in agreement with our recent observation that when the collagen fibrils on cartilage surface undergo OA-like partial degradation, changes in nanoscale aggrecan-collagen molecular adhesion precede changes in histology staining [7]. DMM has been established as a standard method to introduce mild-to-moderate OA [8]. Results from this study will help to better differentiate the acceleration or attenuation of OA in transgenic murine models subjected to DMM [9-12]. Our ongoing studies on the nanomechanical tests at longer time points up to 12 weeks will further elucidate the mechanical deterioration of cartilage during OA progression. At later stages of OA development, nanoindentation may yield higher E ind when the value starts to reflect the properties of cartilage deeper layers and sub-chondral bone upon substantial loss of cartilage. It is imperative to establish a comprehensive understanding of the mechanical symptoms at both early and late stages of osteoarthritis. Significance: We found the decrease in cartilage superficial layer modulus precedes the histological signs of OA after the destabilization of the medial meniscus in mice. This study proposes a new method that can detect the initiation/progression of OA earlier in the widely used DMM model.
ORS 2015 Annual Meeting Paper No: 0125