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.

To investigate the damages to the extracellular matrix in articular cartilage due to cryopreservation, the depth-dependent concentration profiles of glycosaminoglycans (GAGs) in thirty-four cartilage specimens from canine humeral heads were imaged at 13μm pixel resolution using the in vitro version of the dGEMRIC protocol in microscopic MRI (μMRI). In addition, a biochemical assay was used to determine the GAG loss from the tissue to the solution where the tissue was immersed. For specimens that had been frozen at −20 °C or −80 °C without any cryoprotectant, a significant loss of GAG (as high as 56.5%) was found in cartilage, dependent upon the structural zones of the tissue and the conditions of cryopreservation. The cryoprotective abilities of dimethyl sulfoxide (DMSO) as a function of its concentration in saline and storage temperature were also investigated. A 30% DMSO concentration was sufficient in preventing the reduction of GAG in the tissue at the −20 °C storage temperature, but a 50% concentration of DMSO was necessary for the −80 °C cryopreservation. These imaging results were verified by the biochemical analysis.

Since focal stress on the SBP underlying sites of cartilage damage increases during late stages of OA, these findings establish mechanical loading-induced attenuation of sclerostin expression and elevation of bone formation along the SBP surface as the major mechanisms characterizing subchondral bone phenotypes associated with severe late-stage OA in mice.

To investigate the dependency of T1 relaxation on mechanical strain in articular cartilage, quantitative magnetic resonance T1 imaging experiments were carried out on cartilage before/after the tissue was immersed in gadolinium contrast agent and when the tissue was being compressed (up to ∼48% strains). The spatial resolution across the cartilage depth was 17.6 μm. The T1 profile in native tissue (without the presence of gadolinium ions) was strongly strain‐dependent, which is also depth‐dependent. At the modest strains (e.g., 14% strain), T1 reduced by up to 68% in the most surface portion of the tissue. Further compression (e.g., 45% strain) reduced T1 mostly in the middle and deep portions of the tissue. For the gadolinium‐immersed tissue, both modest and heavy compressions (up to 48% strain) increased T1 slightly but significantly, although the overall shapes of the T1 profiles remained approximately the same regardless of the amount of strains. The complex relationships between the T1 profiles and the mechanical strains were a direct consequence of the depth‐dependent proteoglycan concentration in the tissue, which determined the tissue's mechanical properties. This finding has potential implications in the use of gadolinium contrast agent in clinical magnetic resonance imaging of cartilage (the dGEMRIC procedure), when the loading or loading history of patients is considered. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.

The topographical variations of the zonal properties of canine articular cartilage over the medial tibia were evaluated as the function of external loading by microscopic magnetic resonance imaging (µMRI). T2 and T1 relaxation maps and GAG (glycosaminoglycan) images from a total of 70 specimens were obtained with and without the mechanical loading at 17.6 µm depth resolution. In addition, mechanical modulus and water content were measured from the tissue. For the bulk without loading, the means of T2 at magic angle (43.6 ± 8.1 ms), absolute thickness (907.6 ± 187.9 µm) and water content (63.3 ± 9.3%) on the meniscus-covered area were significantly lower than the means of T2 at magic angle (51.1 ± 8.5 ms), absolute thickness (1251.6 ± 218.4 µm) and water content (73.2 ± 5.6%) on the meniscus-uncovered area. However GAG (86.0 ± 15.3 mg/ml) on the covered area was significantly higher than GAG (70.0 ± 8.8 mg/ml) on the uncovered area. Complex relationships were found in the tissue properties as the function of external loading. The tissue parameters in the superficial zone changed more profoundly than the same properties in the radial zone. The tissue parameters in the meniscus-covered areas changed differently when comparing with the same parameters in the uncovered areas. This project confirms that the load-induced changes in the molecular distribution and structure of cartilage are both depth-dependent and topographically distributed. Such detailed knowledge of the tibial layer could improve the early detection of the subtle softening of the cartilage that will eventually lead to the clinical diseases such as osteoarthritis.

To further study the anisotropic distribution of the collagen matrix in articular cartilage, microscopic magnetic resonance imaging experiments were carried out on articular cartilages from the central load-bearing area of three canine humeral heads at 13 mm resolution across the depth of tissue. Quantitative T 2 images were acquired when the tissue blocks were rotated, relative to B 0 , along two orthogonal directions, both perpendicular to the normal axis of the articular surface. The T 2 relaxation rate (R 2 ) was modeled, by three fibril structural configurations (solid cone, funnel, and fan), to represent the anisotropy of the collagen fibrils in cartilage from the articular surface to the cartilage/bone interface. A set of complex and depth-dependent characteristics of collagen distribution was found in articular cartilage. In particular, there were two anisotropic components in the superficial zone and an asymmetrical component in the radial zone of cartilage. A complex model of the three-dimensional fibril architecture in articular cartilage is proposed, which has a leaf-like or layer-like structure in the radial zone, arises in a radial manner from the subchondral bone, spreads and arches passing the isotropic transitional zone, and exhibits two distinct anisotropic components (vertical and transverse) Structural architecture of articular cartilage plays a critical role in the biomechanical functions and morphological properties of the tissue as a load-bearing material in joints (1-6), whose degradation is the hallmark of clinical joint diseases such as osteoarthritis. As the collagen fibril is the principal macromolecule that provides a depth-dependent structural integrity to articular cartilage (7), continuing efforts have been focused on the specific features of the three-dimensional (3D) collagen structure in cartilage. Histologically, the collagen matrix in noncalcified cartilage is commonly considered to contain three structural zones from the articular surface to the cartilage bone interface, namely, the superficial zone (SZ) with the collagen fibrils parallel with the tissue surface, the transitional zone (TZ) with mostly random fibrils, and the radial zone (RZ) with the perpendicular fibrils anchored to the underlining bone. These depth-dependent features of the collagen matrix become the fundamental components in several fibril models in literature, including the arcade model (8), where the collagen fibrils arise in a radial manner from the subchondral bone, pass toward the surface through the TZ obliquely, and return to the bone; the columnar arrangement (9,10), where the collagens are arranged in a columnar manner, that could be traced from the calcified cartilage to their oblique orientation in the tangential tissue matching the concept of the arcade model; and the leaf model (3,5,10,11), where the collagens are arranged in a series of closely packed layers or leaves in the RZ and arches in the TZ to form the horizontally orientated leaves in the SZ.The structural orientation of collagen ...

Fifteen articular cartilage-bone specimens from one canine humeral joint were compressed in the strain range of 0-50%. The deformation of the extracellular matrices in cartilage was preserved and the same tissue sections were studied using polarized light microscopy (PLM) and Fourier-transform infrared imaging (FTIRI). The PLM results show that the most significant changes in the apparent zone thickness due to 'reorganization' of the collagen fibrils based on the birefringence occur between 0% and 20% strain values, where the increase in the superficial zone and decrease in the radial zone thicknesses are approximately linear with the applied strain. The FTIRI anisotropy results show that the two amide components with bond direction perpendicular to the external compression retain anisotropy (amide II in the superficial zone and amide I in the radial zone). In contrast, the measured anisotropy from the two amide components with bond direction parallel to the external compression changes their anisotropy significantly (amide I in the superficial zone and amide II in the radial zone). Statistical analysis shows that there is an excellent correlation (r=0.98) between the relative depth of the minimum retardance in PLM and the relative depth of the amide II anisotropic cross-over. The changes in amide anisotropies in different histological zones are explained by the strain-dependent tipping angle of the amide bonds. These depth-dependent adaptations to static loading in cartilage's morphological structure and chemical distribution could be useful in the future studies of the early diseased cartilage.