Farid Badar

Damages to the extracellular matrix in articular cartilage due to cryopreservation by microscopic magnetic resonance imaging and biochemistry.

To investigate the damages to the extracellular matrix in articular cartilage due to cryopreservation, the depth-dependent concentration profiles of glycosaminoglycans (GAGs) in 34 cartilage specimens from canine humeral heads were imaged at 13-mum pixel resolution using the in vitro version of the dGEMRIC protocol in microscopic MRI (microMRI). 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 degrees C or -80 degrees 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 degrees C storage temperature, but a 50% concentration of DMSO was necessary for the -80 degrees C cryopreservation. These imaging results were verified by the biochemical analysis.

Strain-dependent T1 relaxation profiles in articular cartilage by MRI at microscopic resolutions

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 tissues 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.

Further studies on the anisotropic distribution of collagen in articular cartilage by μMRI

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 μm resolution across the depth of tissue. Quantitative T2 images were acquired when the tissue blocks were rotated, relative to B0, along two orthogonal directions, both perpendicular to the normal axis of the articular surface. The T2 relaxation rate (R2) 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) in the surface portion of the tissue. Magn Reson Med, 2011.

Topographical variations of the strain-dependent zonal properties of tibial articular cartilage by microscopic MRI

Abstract 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.

EGFR signaling is critical for maintaining the superficial layer of articular cartilage and preventing osteoarthritis initiation

Significance The uppermost superficial zone of articular cartilage plays multifaceted roles in maintaining cartilage structure, function, and mechanical properties and in preventing cartilage degeneration during osteoarthritis initiation. However, its regulation by growth factors and hormones is still largely unknown. Here we report that EGFR signaling is an important growth factor pathway that maintains superficial chondrocyte number, promotes boundary lubricant secretion and cartilage surface lubrication, and stimulates mechanical strength of articular cartilage. Reduction in EGFR activity leads to structurally, functionally, and mechanically compromised articular cartilage during development and drastically accelerates cartilage degeneration under normal and surgically induced osteoarthritis conditions. Thus, our studies strongly suggest that targeting cartilage surface EGFR signaling should be considered as a novel direction for osteoarthritis treatment. 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.

Molecular origin of a loading-induced black layer in the deep region of articular cartilage at the magic angle.

To investigate the molecular origin of an unusual low‐intensity layer in the deep region of articular cartilage as seen in magnetic resonance imaging (MRI) when the tissue is imaged under compression and oriented at the magic angle.

Subchondral bone plate sclerosis during late osteoarthritis is caused by loading‐induced reduction in Sclerostin

To establish an unbiased, 3‐dimensional (3‐D) approach that quantifies subchondral bone plate (SBP) changes in mouse joints, and to investigate the mechanism that mediates SBP sclerosis at a late stage of osteoarthritis (OA).

Compressed sensing in quantitative determination of GAG concentration in cartilage by microscopic MRI

To evaluate the potentials of compressed sensing (CS) in MRI quantification of glycosaminoglycan (GAG) concentration in articular cartilage at microscopic resolution.

MRI properties of a unique hypo-intense layer in degraded articular cartilage.

To investigate the characteristics of a hypo-intense laminar appearance in articular cartilage under external loading, microscopic magnetic resonance imaging (μMRI) T1, T2 and T1ρ experiments of a total of 15 specimens of healthy and trypsin-degraded cartilage were performed at different soaking solutions (saline and 100 mM phosphate buffered saline (PBS)). T2 and T1ρ images of the healthy tissue in saline showed no load-induced laminar appearance, while a hypo-intense layer was clearly visible in the deep part of the degraded tissue at the magic angle. A significant difference was found between T2 values at 0° and 55° (from 16.5  ±  2.8 ms to 20.2  ±  2.7 ms, p  =  0.0005), and at 0° and 90° (16.5  ±  2.8 ms to 21.3  ±  2.6 ms, p  <  0.0001) in saline solution. In contrast, this hypo-intense laminar appearance largely disappeared when tissue was soaked in PBS. The visualization of this hypo-intensity appearance in different soaking mediums calls for caution in interpreting the data of relaxation times, chemical exchange and collagen fiber deformation.

Loading-induced changes on topographical distributions of the zonal properties of osteoarthritic tibial cartilage – A study by magnetic resonance imaging at microscopic resolution

The topographical distributions of the zonal properties of articular cartilage over the medial tibia from an experimental osteoarthritis (OA) model were evaluated as a function of external loading by microscopic Magnetic Resonance Imaging (µMRI). T2 relaxation times and cartilage thicknesses were measured at 17.6 µm resolution from 118 specimens, which came from thirteen dogs (six 8-week and seven 12-week after surgery), with and without mechanical loading. In addition, bulk mechanical modulus was measured topographically from each tibia surface. The total thickness decreased significantly under the external loading, in which the relative thickness of the superficial zone (SZ) and the transitional zone (TZ) increased whereas the radial zones (RZs) decreased. In the bulk data, T2(55°) decreased significantly (p<0.001) at all OA-time-points, but T2(0°) decreased without significance (p>0.05) at 8-week. Complex relationships were found in the zonal tissue properties as a function of external loading with the progress of OA. T2 in the superficial zone changed more profoundly than the same properties in the radial zone as a function of external loading at all OA time-points. This study confirms that OA affects the load-induced changes in the molecular distribution and structure of cartilage, which are both depth-dependent and topographically distributed. Such detailed knowledge of mechanobiological changes in specific tibial cartilage zones and locations with OA progress could improve the early detection of the subtle softening of cartilage that accompanies pre-clinical stages of OA.