Objective This study aims to demonstrate that cartilage nanoindentation modulus is a highly sensitive indicator of the onset and spatiotemporal progression of post-traumatic osteoarthritis (PTOA) in murine models. Design Destabilization of medial meniscus (DMM) surgery was performed on the right knees of 12-week old male, wild-type C57BL/6 mice, with Sham control on contralateral left knees. Atomic force microscopy (AFM)-based nanoindentation was applied to quantify the nanoindentation modulus, Eind, of femoral condyle cartilage at 3 days to 12 weeks after surgery. The modulus changes were compared against the timeline of histological OA signs. Meanwhile, at 8 weeks after surgery, changes in meniscus, synovium and subchondral bone were evaluated to reveal the spatial progression of PTOA. Results The modulus of medial condyle cartilage was significantly reduced at 1 week after DMM, preceding the histological OA signs, which only become detectable at 4 – 8 weeks after. This reduction is likely due to concomitantly elevated proteolytic activities, as blocking enzymatic activities in mice can attenuate this modulus reduction. In later OA, lateral condyle cartilage and medial meniscus also started to be weakened, illustrating the whole-organ nature of PTOA. Conclusions This study underscores the high sensitivity of nanoindentation in examining the initiation, attenuation and progression of PTOA in murine model. Meanwhile, modulus changes highlight concomitant changes in lateral cartilage and meniscus during the advancement of OA.

Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.EGFR | articular cartilage | chondrocyte | lubrication | osteoarthritis

ganglion (DRG) sensory neurons, including >90% of C-nociceptors (pain-sensing neurons) and C-low-threshold mechanoreceptors, as well as a lower percentage of Ad-nociceptors and Ab afferents. At age 10 weeks (n ¼ 5) and at age 26 weeks (n ¼ 5), mice were perfused transcardially with paraformaldehyde, and the right knees were collected, post fixed and decalcified. Twenty-mm thick frozen sections were collected at mid-joint level. Consecutive sections were stained with hematoxylin & eosin. Age-matched heterozygous C57BL/6 Pirt-GCaMP3 mice were used to confirm innervation patterns. These mice express the green fluorescent calcium indicator, GCaMP3, in~90% of all sensory DRG neurons (including the Na v 1.8 population), and not in other peripheral or central tissues, through the Pirt promoter. Results: Examination of the knees of 10-week old Na V 1.8-TdTomato mice revealed areas of dense innervation by Na V 1.8-expressing sensory fibers, most notably the bone marrow, the lateral synovium, and the connective tissue layer (epiligament) surrounding the cruciate ligaments, including the areas of attachment. Other structures, such as the medial synovium and the collagenous substance of the cruciate ligaments, were less densely innervated. Na V 1.8 nociceptors were also present in the outer third of the lateral meniscus. The articular cartilage, the inner two thirds of the lateral meniscus, and the medial meniscus did not show innervation. Figure 1 shows an example of these features in one mouse-but these findings were remarkably reproducible in n ¼ 5 mice. Assessment of Na V 1.8 signal in knees of 26-week old mice revealed marked changes in innervation density (not shown). Compared to 10-week old knees, 26-week old knees showed a dramatic decline in Na V 1.8-expressing nociceptors in the lateral synovium, as well as in the epiligament and attachment areas of the cruciate ligaments. Similar age-related changes in the innervation were also detected in the knees of 26-week old Pirt-GCaMP3 mice compared to 10-week old knees, providing independent evidence that the chosen markers are specific for nerve fibers. Conclusions: This study reproducibly shows, for the first time, that the nociceptive innervation of specific murine knee tissues dramatically declines with age. Remarkably, this occurs quite early on in the life of the mouse, where we find dense innervation at 10 weeks and a marked decline by 26 weeks. Ongoing studies are aimed at monitoring innervation with more advanced age. The biological significance of these findings needs to be explored, as well as the relationship with pathogenesis of osteoarthritis.

Joint biomechanical functions rely on the integrity of cartilage extracellular matrix. Understanding the molecular activities that govern cartilage matrix assembly is critical for developing effective cartilage regeneration strategies. This study elucidated the role of decorin, a small leucine-rich proteoglycan, in the structure and biomechanical functions of cartilage. In decorinnull cartilage, we discovered a substantial reduction of aggrecan content, the major proteoglycan of cartilage matrix, and mild changes in collagen fibril nanostructure. This loss of aggrecan resulted in significantly impaired biomechanical properties of cartilage, including decreased modulus, elevated hydraulic permeability, and reduced energy dissipation capabilities. At the cellular level, we found that decorin functions to increase the retention of aggrecan in the neo-matrix of chondrocytes, rather than to directly influence the biosynthesis of aggrecan. At the molecular level, we demonstrated that decorin significantly increases the adhesion between aggrecan and aggrecan molecules and between aggrecan molecules and collagen II fibrils. We hypothesize that decorin plays a crucial structural role in mediating the matrix integrity and biomechanical functions of cartilage by providing physical linkages to increase the adhesion and assembly of aggrecan molecules at the nanoscale.

This study aimed to quantify the biomechanical properties of murine meniscus surface. Atomic force microscopy (AFM)-based nanoindentation was performed on the central region, proximal side of menisci from 6-to 24-week old male C57BL/6 mice using microspherical tips (R tip ≈ 5 μm) in PBS. A unique, linear correlation between indentation depth, D, and response force, F, was found on menisci from all age groups. This non-Hertzian behavior is likely due to the dominance of tensile resistance by the collagen fibril bundles on meniscus surface that are mostly aligned © 2015 Published by Elsevier Ltd. *Correspondence and requests for materials should be addressed to: Dr. Lin Han Phone: (215)571-3821 Fax: (215)895-4983 lh535@drexel.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of interest statementThe authors of this study have no personal or financial conflicts of interest with this work. All authors were fully involved in the study and preparation of this manuscript and the material within has not been and will not be submitted for publication elsewhere. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript along the circumferential direction observed on 12-week old menisci. The indentation resistance was calculated as both the effective stiffness, S ind = dF/dD, and the effective modulus, E ind , via the isotropic Hertz model. Values of S ind and E ind were found to depend on indentation rate, suggesting the existence of poro-viscoelasticity. These values do not significantly vary with anatomical sites, lateral versus medial compartments, or mouse age. In addition, E ind of meniscus surface (e.g., 6.1 ± 0.8 MPa for 12 weeks of age, mean ± SEM, n = 13) was found to be significantly higher than those of meniscus surfaces in other species, and of murine articular cartilage surface (1.4 ± 0.1 MPa, n = 6). In summary, these results provided the first direct mechanical knowledge of murine knee meniscus tissues. We expect this understanding to serve as a mechanics-based benchmark for further probing the developmental biology and osteoarthritis symptoms of meniscus in various murine models.