Assessment of post-trauma microstructural alterations in the rabbit knee cartilage and subchondral bone.

Early diagnosis of post-traumatic osteoarthritis (PTOA) is critical for designing better treatments before the degradation becomes irreversible. We utilized multimodal high-resolution imaging to investigate early-stage deterioration in articular cartilage and the subchondral bone plate from a sub-critical impact to the knee joint, which initiates PTOA. The knee joints of 12 adult rabbits were mechanically impacted once on the femoral articular surface to initiate deterioration. At 2- and 14-week post-impact surgery, cartilage-bone blocks were harvested from the impact region in the animals (N = 6 each). These blocks were assessed for deterioration using polarized light microscopy (PLM), microcomputed tomography (µCT), and biochemical analysis. Statistically significant changes were noted in the impact tissues across the calcified zone (CZ) at 14 weeks post-impact: the optical retardation values in the CZ of impact cartilage had a drop of 29.0% at 14 weeks, while the calcium concentration in the CZ of impact cartilage also had a significant drop at 14 weeks. A significant reduction of 6.3% in bone mineral density (BMD) was noted in the subchondral bone plate of the impact samples at 14 weeks. At 2 weeks post-impact, only minor, non-significant changes were measured. Furthermore, the impact knees after 14 weeks had greater structural changes compared with the 2-week impact knees, indicating progressive degradation over time. The findings of this study facilitated a connection between mineralization alterations and the early deterioration of knee cartilage after a mechanical injury. In a broader context, these findings can be beneficial in improving clinical strategies to manage joint injuries.

Assessment of articular cartilage degradation in response to an impact injury using µMRI.

PURPOSE: Degradation of articular cartilage (AC) due to injury to the knee joint may initiate post-traumatic osteoarthritis (PTOA). Failure to diagnose the onset of the disease at an early stage makes the cure ineffective for PTOA. This study investigated the consequences of a mechanical injury to the knee in a rabbit model using microscopic magnetic resonance imaging (µMRI) at high resolution. MATERIALS AND METHODS: A mechanical injury was induced to the knee joints of 12 rabbits. Cartilage blocks were extracted from the non-impacted and impacted knee joints after 2 and 14 weeks post-impact. The specimens were studied using µMRI T2 relaxation and inductively coupled plasma analysis to determine the early degradation of the articular cartilage. RESULTS: The data established a connection between T2 relaxation time and the early progression of knee PTOA after an impact injury. T2 values were found to be higher in the impacted cartilage at both 2 and 14 weeks, in particular, T2-55° values in the impacted samples displayed a significant rise of 6.93% after 2 weeks and 20.02% after 14 weeks. Lower glycosaminoglycan measurement and higher water content in the impacted cartilage confirmed the µMRI results. CONCLUSIONS: This µMRI T2 study was able to detect cartilage damage in the impacted knees. In addition, greater degradation in the affected knees at 14 weeks than at 2 weeks indicated the progressive nature of cartilage deterioration over time. The µMRI results were in accord with the biochemical analysis, indicating the detection of early structural damage in the cartilage.

Study of the collagen tissue nanostructure by analyzing the echo decay obtained using the MRI technique.

The multicomponent relaxation observed in nuclear magnetic resonance experiments in biological tissues makes it difficult to establish a correlation between specific relaxation times and tissue structural parameters. The analysis of a nanostructure (the characteristic size of 10-1000 nm) is usually based on formulas for relaxation times which depend on structural parameters at the atomic or molecular levels in the size range of 0.1-5 nm. We have recently developed an analysis method in which relaxation times' anisotropy in a sample is explicitly related to its structure of nanocavities containing a liquid or gas. However, the method is based on the analysis of experimental data on the anisotropy of relaxation times obtained by using the standard NMR technique and rotating the sample relative to a magnetic field and requires a series of experiments. In the present study, to address this challenge, we develop a new method of analysis of a multi-exponential magnetic resonance signal that does not require determining relaxation times and eliminates the sample rotation and the necessity of a series of experiments. Using the magnetic resonance imaging (MRI) technique, the total signal from the whole sample was obtained as a sum of the signals (echo decays) from all voxels. In contrast to previous research, the volumes of nanocavities and their angular distribution can be obtained by analyzing a single total signal for the entire cartilage. In addition, within the framework of this approach, it is possible to identify the reason for the multicomponent nature of relaxation. The proposed method for analyzing a single multi-exponential signal (transverse relaxation) was implemented on cartilage. Using the signal, three anatomical zones of cartilage were studied, revealing significant structural differences between them. The proposed method not only avoids the need for sample rotation but also enables repeated application of layer-by-layer magnetic resonance imaging with micron resolution. The study results allow us to suggest that water molecules contributing to the echo decay are more likely located in nanocavities formed by the fibrillar structure rather than inside the fibrils.

Structural differences between immature and mature articular cartilage of rabbits by microscopic MRI and polarized light microscopy.

This study aimed to determine the structural features between immature and mature articular cartilage from the humeral and femoral joints of rabbits. Specimens of articular cartilage (n = 6 for immature tissue, n = 6 for mature tissue) that were still attached to the underlying bone from a humerus (shoulder joint) or femur (knee joint) were imaged using microscopic MRI (µMRI) and polarized light microscopy (PLM). Quantitative µMRI data with a pixel resolution of 11.7-13.2 µm revealed a number of differences between the immature and mature cartilage, including total thickness, and T2 and T1ρ relaxation values. Quantitative PLM data with a pixel resolution of 0.25-1 µm confirmed the µMRI results and revealed additional differences in cellular features between the tissues. The mature cartilage had a clearly defined tidemark, which was absent in the immature tissue. The ability to differentiate specific maturation-related cartilage characteristics could be beneficial to translational studies of degenerative diseases such as osteoarthritis.

Resolution-dependent influences of compressed sensing in quantitative T2 mapping of articular cartilage.

This study evaluates the resolution-dependent influences of compressed sensing (CS) in MRI quantification of T2 mapping in articular cartilage with osteoarthritis (OA). T2-weighed 2D experiments of healthy and OA cartilage were fully sampled in k-space with five echo times at both 17.6 µm and 195.3 µm in-plane resolutions; termed as microscopic MRI (µMRI) and macroscopic MRI (mMRI) respectively. These fully sampled k-space data were under-sampled at various 2D CS accelerating factors (AF = 4-32). The under-sampled data were reconstructed individually into 2D images using nonlinear reconstruction, which were used to calculate the T2 maps. The bulk and zonal variations of T2 values in cartilage were evaluated at different AFs. The study finds that the T2 images at AFs up to 8 preserved major visual information and produced negligible artifacts for µMRI. The T2 values remained accurate for different sub-tissue zones at various AFs. The absolute difference between the CS (AF up to 32) and the Ground Truth (i.e., using 100% of the k-space data) of the mean T2 values through the whole tissue depth was higher in mMRI versus µMRI. For mMRI (where the resolution mimics the clinical MRI of human cartilage), the quantitative T2 mapping at AFs up to 4 showed negligible variations. This study demonstrates that both clinical MRI and µMRI can benefit from the use of CS in image acquisition, and µMRI benefits more from the use of CS by acquiring much less data, without losing significant accuracy in the quantification of T2 maps in osteoarthritic cartilage.

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

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.

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

PURPOSE: To evaluate the potentials of compressed sensing (CS) in MRI quantification of glycosaminoglycan (GAG) concentration in articular cartilage at microscopic resolution. METHODS: T1 -weighted 2D experiments of cartilage were fully sampled in k-space with five inversion times at 17.6 µm resolution. These fully sampled k-space data were re-processed, by undersampling at various 1D and 2D CS undersampling factors (UFs). The undersampled data were reconstructed individually into 2D images using nonlinear reconstruction, which were used to calculate 2D maps of T1 and GAG concentration. The values of T1 and GAG in cartilage were evaluated at different UFs (up to 16, which used 6.25% of the data). K-space sampling pattern and zonal variations were also investigated. RESULTS: Using 2D variable density sampling pattern, the T1 images at UFs up to eight preserved major visual information and produced negligible artifacts. The GAG concentration remained accurate for different sub-tissue zones at various UFs. The variation of the mean GAG concentration through the whole tissue depth was 1.20%, compared to the fully sampled results. The maximum variation was 2.24% in the deep zone of cartilage. Using 1D variable density sampling pattern, the quantitative T1 mapping and GAG concentration at UFs up to 4 showed negligible variations. CONCLUSION: This study demonstrates that CS could be beneficial in microscopic MRI (µMRI) studies of cartilage by acquiring less data, without losing significant accuracy in the quantification of GAG concentration. Magn Reson Med 79:3163-3171, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Topographical variations in zonal properties of canine tibial articular cartilage due to early osteoarthritis: a study using 7-T magnetic resonance imaging at microscopic resolution.

OBJECTIVE: Our aim was to determine topographical variations in zonal properties of articular cartilage over the medial tibia in an experimental osteoarthritis (OA) model using 7-T magnetic resonance imaging (MRI). MATERIALS AND METHODS: An anterior cruciate ligament (ACL)-transection canine model was subjected to study at 8 (six) and 12 (seven) weeks after the surgery. Each medial tibia was divided into five topographical locations. For each specimen, T2 relaxation (at 0° and 55°) was quantified at microscopic resolution. The imaging data grouped the five locations into two topographical areas (meniscus-covered and -uncovered). RESULTS: The T2 (55°) bulk values from the meniscus-covered area were significantly lower than those from the uncovered area. The total cartilage thicknesses on the meniscus-covered area were significantly thinner than those on the meniscus-uncovered area. Significant differences in the T2 (0°) values were observed in most thicknesses of the four subtissue zones and whole-tissue from the uncovered area, while the same significant changes were detected in the superficial zone from the meniscus-covered area. CONCLUSION: By quantifying high-resolution imaging data both topographically and depth-dependently (zonal-wise), this study demonstrates that the rate of disease progression varies topographically over the medial tibia. Future correlation with OA pathology could lead to better detection of early OA.

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

PURPOSE: 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. MATERIALS AND METHODS: Microscopic MRI (µMRI) T2 and T1 ρ experiments were carried out on 18 specimens, both native and degraded (treated with trypsin). The glycosaminoglycan (GAG) concentrations in the specimens were quantified by both sodium ICP-OES and µMRI Gd(DTPA)(2-) -contrast methods. The mechanical modulus of the specimens was also measured. RESULTS: Native tissue shows no load-induced layer, while the trypsin-degraded tissue shows clearly the low-intensity line at the deep part of tissue. The GAG reductions were confirmed by the sodium ICP-OES (from 81.7 ± 5.4 mg/mL to 9.2 ± 3.4 mg/mL), MRI GAG quantification (from 72.4 ± 6.7 mg/mL to 11.2 ± 2.9 mg/mL). The modulus reduction was confirmed by biomechanics (from 4.3 ± 0.7 MPa to 0.3 ± 0.1 MPa). CONCLUSION: Both T2 and T1 ρ profiles in native and degraded cartilage show strongly strain-, depth-, and angle-dependence using high-resolution MRI. The GAG reduction is responsible for the visualization of a low-intensity layer in deep cartilage when it is loaded and oriented at 55°.

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

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.

Characteristics of distal femoral articular cartilage in 6 weeks posttraumatic osteoarthritis by a subcritical impact.

Traumatized knee greatly contributes to osteoarthritis (OA) of the knee in young adults. To intervene effectively before the onset of severe structural disruption, detection of the disease at the early onset is crucial. In this study, we put together the findings for the detection of OA from the femoral knee joint cartilage of the rabbit at 6 weeks posttrauma. Articular cartilage samples are taken from the impacted and nonimpacted joints at 0 week (serving as the control group) and at 6 weeks posttrauma by minimal force. The samples were imaged using microscopic magnetic resonance imaging (µMRI) at 11.7 µm/pixel and polarized light microscopy (PLM) at 1 µm/pixel. In addition, an inductively coupled plasma - optical emission spectrometry analysis was performed using the adjacent cartilage samples. The outcomes of this study demonstrate an increase in T2 values in 6 weeks samples compared to the 0 week samples by µMRI technique, indicating a general increase of tissue hydration within cartilage. PLM detects a decrease in the average thickness of the superficial zones in the posttraumatic osteoarthritis samples, significant in the impacted femurs. There was an average increasing trend of maximum retardation in the tide mark in comparison to the reported calcium concentration (mg/L) in impacted samples suggesting a possible rise in mineralization in the 6 weeks samples. Qualitatively, physical observation of the joint after 6 weeks showed signs of reddening in the anterior femur suggesting the disease process is a localized phenomenon. Through microscopic imaging, we are able to detect these changes at 6 weeks posttrauma qualitatively and quantitatively.

The interface region between articular cartilage and bone by µMRI and PLM at microscopic resolutions.

This dual-modality microscopic imaging study quantifies the interface region between the noncalcified cartilage and the subchondral bone plate, which includes the deep portion of the noncalcified articular cartilage and the zone of calcified cartilage (ZCC). This interface region is typically not visible in routine MRI but becomes visible in MRI with the application of an ultra-short echo time (UTE) sequence. A number of cartilage-bone blocks from a well-documented canine humeral head were harvested for imaging by microscopic MRI (µMRI) and PLM (polarized light microscopy). In µMRI, T2 anisotropic images were acquired by 2D gradient-echo, magnetization-prepared spin-echo and UTE sequences at the 0° and 55° (the magic angle) orientations at 11.7 µm/pixel resolution. In PLM, quantitative optical retardation (nm) and collagen orientation (°) were mapped from the thin sections from the same µMRI specimens at 0.5-2 µm pixel resolutions. The orientational and organizational architecture of the collagen matrix in this interface region was quantified and correlated between the complementary imaging. The magic angle effect as seen in the noncalcified cartilage was statistically confirmed in ZCC in µMRI, which was further supported by quantitative PLM. With an enhanced understanding of the tissue properties in this important interface region, it will potentially be possible to monitor the changes of this tissue region which is instrumental to the initiation and development of osteoarthritis and other joint diseases.

Location-Specific Study of Young Rabbit Femoral Cartilage by Quantitative µMRI and Polarized Light Microscopy.

OBJECTIVE: Microscopic magnetic resonance imaging (µMRI) and polarized light microscopy (PLM) are used to characterize the structural variations at different anatomical locations of femoral cartilage in young rabbits (12-14 weeks old). DESIGN: Four intact knees were imaged by µMRI at 86 µm resolution. Three small cartilage-bone specimens were harvested from each of 2 femoral medial condyles and imaged by quantitative µMRI (T2 anisotropy) at 9.75 µm resolution (N = 6). These specimens, as well as the other 2 intact femoral condyles, were used for histology and imaged by quantitative PLM (retardation and angle) at 0.25 µm to 4 µm resolutions. RESULTS: Quantitative MRI relaxation data and PLM fibril data revealed collaboratively distinct topographical variations in both cartilage thickness and its collagen organization in the juvenile joint. Cartilage characteristics from the central location have a 3-zone arcade-like fibril structure and a distinct magic angle effect, commonly seen in mature articular cartilage, while cartilage at the anterior location lacks these characteristics. Overall, the lowest retardation values and isotropic T2 values have been found in the distal femur (trochlear ridge), with predominant parallel fibers with respect to the articular surface. Central cartilage is the thickest (~550 µm), approximately twice as thick as the anterior and posterior locations. CONCLUSION: Distinctly different characteristics of tissue properties were found in cartilage at different topographical locations on femoral condyle in rabbits. Knowledge of location-specific structural differences in the collagen network over the joint surface can improve the understanding of local mechanobiology and provide insights to tissue engineering and degradation repairs.

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.

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

To investigate the dependency of T(1) relaxation on mechanical strain in articular cartilage, quantitative magnetic resonance T(1) 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 T(1) 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), T(1) reduced by up to 68% in the most surface portion of the tissue. Further compression (e.g., 45% strain) reduced T(1) 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 T(1) slightly but significantly, although the overall shapes of the T(1) profiles remained approximately the same regardless of the amount of strains. The complex relationships between the T(1) 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.

Topographical and zonal patterns of T2 relaxation in osteoarthritic tibial cartilage by low- and high-resolution MRI.

PURPOSE: This study aimed to establish the topographical and zonal T2 patterns of multi-resolution MRI in medial tibial cartilage in a canine model of osteoarthritis (OA), initiated by the anterior cruciate ligament (ACL) transection surgery, and studied after 8-weeks and 12-weeks post-surgery. METHODS: Articular cartilage from healthy, two stages of contralateral, and of OA knees were quantitatively imaged by the MRI T2 protocols at two imaging resolutions (100 and 17.6 µm/pixel). The zonal T2 changes at five topographical locations (anterior (AMT), exterior (EMT), posterior (PMT), central (CMT) and interior (IMT) medial tibia) and subsequent two averaged regions (covered by meniscus and exposed) were analyzed. At each location, full-thickness cartilage was studied in four sub-tissue zones (superficial, transitional, upper and lower radial zones). RESULTS: Tissue degradation can be detected by measurable changes of T2, which is resolution- and orientation-dependent. T2 changes ranging from +28.82% increase (SZ, PMT) to -23.15% decrease (RZ1, AMT) in healthy to disease (8C), with the largest increase of T2 in the surface tissue. Various location-dependent patterns of degradation are found over the tibial surface, most commonly shown in early-stage OA (8C) on the anterior site, different from the posterior. Finally, the contralateral cartilage has specific degradation patterns, different from those in OA cartilage. CONCLUSIONS: This is the first quantitative and highest multi-resolution characterization of cartilage at five topographical locations over the medial tibial plateau with fine zonal resolution in an animal model of OA, which would benefit future investigation of human OA in clinics.

Quantitative µMRI and PLM study of rabbit humeral and femoral head cartilage at sub-10 µm resolutions.

This study aimed to establish the baseline characteristics in humeral and femoral cartilage in rabbit, using quantitative magnetic resonance imaging (MRI) relaxation times (T2, T1ρ, and T1) at 9.75 and 70-82 µm pixel resolutions, and quantitative polarized light microscopy (PLM) measures (retardation, angle) at 1.0 and 4.0 µm pixel resolutions. Five intact (i.e., unopened) shoulder joints (the scapula and humeral heads) and three femoral heads of the hip joints from five healthy rabbits were imaged in MRI at 70-82 µm resolution. Thirteen cartilage-bone specimens were harvested from these joints and imaged in µMRI at 9.75 µm resolution. Subsequently, quantitative PLM study of these specimens enabled the examination of the fibril orientation and organization in both intact joints and individual specimens. Quantitative MRI relaxation data and PLM fibril structural data show distinct features in tissue properties at different depths of cartilage, different in individual histological zones. The thicknesses of the histological zones in µMRI and PLM were successfully obtained. This is the first correlated and quantitative MRI and PLM study of rabbit cartilage at sub-10 µm resolutions, which benefits future investigation of osteoarthritis using the rabbit model. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1052-1062, 2020.

Experimental Influences in the Accurate Measurement of Cartilage Thickness in MRI.

OBJECTIVE: To study the experimental influences to the measurement of cartilage thickness by magnetic resonance imaging (MRI). DESIGN: The complete thicknesses of healthy and trypsin-degraded cartilage were measured at high-resolution MRI under different conditions, using two intensity-based imaging sequences (ultra-short echo [UTE] and multislice-multiecho [MSME]) and 3 quantitative relaxation imaging sequences (T1, T2, and T1ρ). Other variables included different orientations in the magnet, 2 soaking solutions (saline and phosphate buffered saline [PBS]), and external loading. RESULTS: With cartilage soaked in saline, UTE and T1 methods yielded complete and consistent measurement of cartilage thickness, while the thickness measurement by T2, T1ρ, and MSME methods were orientation dependent. The effect of external loading on cartilage thickness is also sequence and orientation dependent. All variations in cartilage thickness in MRI could be eliminated with the use of a 100 mM PBS or imaged by UTE sequence. CONCLUSIONS: The appearance of articular cartilage and the measurement accuracy of cartilage thickness in MRI can be influenced by a number of experimental factors in ex vivo MRI, from the use of various pulse sequences and soaking solutions to the health of the tissue. T2-based imaging sequence, both proton-intensity sequence and quantitative relaxation sequence, similarly produced the largest variations. With adequate resolution, the accurate measurement of whole cartilage tissue in clinical MRI could be utilized to detect differences between healthy and osteoarthritic cartilage after compression.

Quantitative measurement of T2, T1ρ and T1 relaxation times in articular cartilage and cartilage-bone interface by SE and UTE imaging at microscopic resolution.

Both spin-echo (SE) and ultra-short echo (UTE) based MRI sequences were used on a 7 T µMRI system to quantify T2, T1ρ and T1 relaxation times from articular cartilage to the cartilage-bone interface on canine humeral specimens at 19.5 µm pixel resolution. A series of five relaxation-weighted images were acquired to calculate one relaxation map (T2, T1ρ or T1), from which the depth-dependent profiles were examined between the SE method and the UTE method, over the entire non-calcified cartilage and within the cartilage-bone interface. SE-based methods enabled the quantification of relaxation profiles over the noncalcified cartilage, from 0 µm (articular surface) to approximately 460 µm in depth (near the end of radial zone). Most of the cartilage-bone interface was imaged by the UTE-based methods, to a tissue depth of about 810 µm. Pixel-by-pixel calculation of the relaxation times between the independent SE and UTE methods correlated well with each other. A better understanding of the tissue properties reliably over the cartilage-bone interface region by a non-invasive MRI approach could contribute to the clinical diagnostics of trauma-induced osteoarthritis.

Loading-Induced Reduction in Sclerostin as a Mechanism of Subchondral Bone Plate Sclerosis in Mouse Knee Joints During Late-Stage Osteoarthritis.

OBJECTIVE: 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). METHODS: A new micro-computed tomography (micro-CT) protocol was developed to characterize the entire thickness of the SBP in the distal femur of a normal mouse knee. Four mouse models of severe joint OA were generated: cartilage-specific Egfr-knockout (Egfr-CKO) mice at 2 months after surgical destabilization of the medial meniscus (DMM), Egfr-CKO mice with aging-related spontaneous OA, wild-type (WT) mice at 10 months after DMM, and WT mice at 14 weeks after DMM plus hemisectomy of the meniscus (DMMH) surgery. As an additional model, mice with knockout of the sclerostin gene (Sost-KO) were subjected to DMMH surgery. Knee joints were examined by micro-CT, histology, and immunohistochemical analyses. RESULTS: Examination of the mouse distal femur by 3-D micro-CT revealed a positive correlation between SBP thickness and the loading status in normal knees. In all 4 mouse models of late-stage OA, SBP sclerosis was restricted to the areas under severely eroded articular cartilage. This was accompanied by elevated bone formation at the bone marrow side of the SBP and a drastic reduction in the levels of sclerostin in osteocytes within the SBP. Unlike in WT mice, no further increase in the thickness of the SBP was observed in response to DMMH in Sost-KO mice. CONCLUSION: 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.