Collagen Fibers In Articular Cartilage Research Articles

T 2 orientation anisotropy mapping of articular cartilage using qMRI

Objective. To provide orientation-independent MR parameters potentially sensitive to articular cartilage degeneration by measuring isotropic and anisotropic components of T 2 relaxation, as well as 3D fiber orientation angle and anisotropy via multi-orientation MR scans. Approach. Seven bovine osteochondral plugs were scanned with a high angular resolution of thirty-seven orientations spanning 180° at 9.4 T. The obtained data was fitted to the magic angle model of anisotropic T 2 relaxation to produce pixel-wise maps of the parameters of interest. Quantitative Polarized Light Microscopy (qPLM) was used as a reference method for the anisotropy and fiber orientation. Main results. The number of scanned orientations was found to be sufficient for estimating both fiber orientation and anisotropy maps. The relaxation anisotropy maps demonstrated a high correspondence with qPLM reference measurements of the collagen anisotropy of the samples. The scans also enabled calculating orientation-independent T 2 maps. Little spatial variation was observed in the isotropic component of T 2 while the anisotropic component was much faster in the deep radial zone of cartilage. The estimated fiber orientation spanned the expected 0°–90° in samples that had a sufficiently thick superficial layer. The orientation-independent magnetic resonance imaging (MRI) measures can potentially reflect the true properties of articular cartilage more precisely and robustly. Significance. The methods presented in this study will likely improve the specificity of cartilage qMRI by allowing the assessment of the physical properties such as orientation and anisotropy of collagen fibers in articular cartilage.

Change in Susceptibility Values in Knee Cartilage After Marathon Running Measured Using Quantitative Susceptibility Mapping.

Quantitative susceptibility mapping (QSM) has been used to study the magnetic susceptibility properties of collagen fibers in articular cartilage; however, it is unclear whether QSM is sensitive to changes due to degradation caused by long-distance running. It is clinically important to understand the link between long-distance running and microstructural changes in knee cartilage. To investigate the ability of QSM to assess microstructural changes within cartilage after repetitive loading. Prospective. Thirteen recreational, male long-distance runners. Three-dimensional gradient recalled echo acquired at 3 T. Magnetic resonance imaging (MRI) and 3D kinematics (translations and rotations during treadmill walking and running) of the knee joint were collected before and after marathon running. The compartments for analysis included the patella, trochlea, and subregions of femoral and tibial cartilage. Changes in regional susceptibility and cartilage thickness were calculated after marathon running. A susceptibility profile was obtained by fitting susceptibility as a function of the normalized depth of cartilage from the superficial to deep layers. Paired t-test or Wilcoxon signed-rank test, 95% confidence interval (CI) of the depth-wise susceptibility profile, Pearson correlation or Spearman correlation. There was a statistically significant increase in susceptibility value in the weight-bearing region of central medial femoral cartilage (cMF-c) after marathon running (pre-marathon: -0.0219 ± 0.0151 ppm, post-marathon: -0.0070 ± 0.0213 ppm, P < 0.05), while the cartilage thickness did not show significant changes in any regions (P-value range: 0.068-0.963). Significant susceptibility elevations occurred in the middle and deep layers of cMF-c (95% CIs did not overlap). A trend toward a positive correlation was found between the changes in susceptibility value in cMF-c and proximal-distal translation of the knee joint during walking (r = 0.55, P = 0.101) and running (r = 0.57, P = 0.089). Localized magnetic susceptibility alterations were observed within knee cartilage in the weight-bearing area after repetitive loading without any morphologic changes. 2 TECHNICAL EFFICACY: Stage 2.

Assessment of collagen fiber orientation dispersion in articular cartilage by small-angle X-ray scattering and diffusion tensor imaging: Preliminary results

Measurements of the orientational dispersion of collagen fibers in articular cartilage were made using diffusion tensor imaging (DTI) and small-angle X-ray scattering (SAXS) on matched bovine articular cartilage samples. Thirteen pairs of samples were excised from bovine knee joints; each pair was taken from neighboring locations in the same bone. One sample from each pair was used for DTI measurements and the other for SAXS measurements. Fractional anisotropy (FA) values were calculated from the DTI data both for the individual imaging voxels and for whole regions of interest (ROI). The FA values were used as a measure of fiber dispersion and compared to the ellipticities of the fiber orientation distributions obtained from SAXS. Neither the spatially-resolved FA values nor whole-ROI FA values showed any correlation with SAXS ellipticities. We attribute the lack of DTI-SAXS correlation to two principal factors: (1) the significant difference in the imaging resolution of the two techniques; and (2) the inherent limitations of both the SAXS data analysis methodology and the diffusion tensor model in the case of multi-modal fiber orientation distributions. We discuss how these factors could be overcome in future work.

Conical scan polarization-sensitive optical coherence tomography

We report on a new articular cartilage imaging technique with potential for clinical arthroscopic use, by supplementing the variable-incidence-angle polarization-sensitive optical coherence tomography method previously developed by us with a conical beam scan protocol. The technique is validated on bovine tendon by comparing experimental data with simulated data generated using the extended Jones matrix calculus. A unique capability of this new optical technique is that it can locate the "brushing direction" of collagen fibers in articular cartilage, which is structural information that extends beyond established methods such as split-line photography or birefringent fast-axis measurement in that it is uniquely defined over the full azimuthal-angle range of (-π, + π). The mapping of this direction over the cartilage surface may offer insights into the optimal design of tissue-engineering scaffolds for cartilage repair.

Experimental validation of an extended Jones matrix calculus model to study the 3D structural orientation of the collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography

We report results to verify a theoretical framework to analyze the 3D depth-wise structural organization of collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography. Apparent birefringence data obtained from multi-angle measurements using a time domain polarization-sensitive optical coherence tomography system has been compared with simulated data based on the extended Jones matrix calculus. Experimental data has been shown to agree with the lamellar model previously proposed for the cartilage microstructure based on scanning electron microscopy data. This tool could have potential application in mapping the collagen structural orientation information of cartilage non-invasively during arthroscopy.

Multinuclear NMR and microscopic MRI studies of the articular cartilage nanostructure

Studies of the structure of articular cartilage by a number of NMR spectroscopic and imaging techniques are reviewed. Advantage is taken of the fact that the NMR investigations can be done non-invasively on the intact tissue and do not require sectioning, slicing and decalcification as in the case of electron microscopy. The different contributions to 1H T2 relaxation are described and it is pointed out that ignoring the biexponential behavior of the transverse relaxation can lead to serious errors in the proton density measurements and the T2 characterization of the articular cartilage. A way to slow the transverse relaxation and to minimize its angular dependence by the use of dipolar echo is described. 2H double quantum filtered spectroscopic MRI is a powerful technique to follow the orientation and density of the collagen fibers in articular cartilage. Using this technique, it was found that attachment of the cartilage to the bone has a stabilizing effect on the collagen matrix and that the hydroxyapatite in the calcified zone resides near the collagen fibers but does not contribute to their order. In response to mechanical pressure, it was shown that the collagen fibers flatten near the surface and become crimped near the bone. A number of NMR techniques have been described for the measurement of 23Na residual quadrupolar interaction. It was found that this can serve as a very sensitive measure of the depletion of proteoglycans. Finally, a combination of the above techniques was used to study a maturation of articular cartilage in pigs. The increased order and density of the collagen fibers from newborn to adult pigs revealed itself as a shortening of T2 and significant increase of the residual quadrupolar interaction of both 2H and 23Na nuclei.

The effect of decalcification on the microstructure of articular cartilage assessed by 2H double quantum filtered spectroscopic MRI

2H quadrupolar splitting of deuterated water molecules is a sensitive measure of the order and density of the collagen fibers in articular cartilage. In the calcified zone, near the bone, two pairs of quadrupolar split satellites were previously observed. To examine whether the large splitting observed originates from the presence of calcium ions and hydroxyapatite, one-dimensional 2H single and double quantum filtered spectroscopic imaging were performed on articular cartilage-bone plugs before and after decalcification. After decalcification, the magnitude splitting of the two pairs of satellites did not change and orientation dependency was kept. However, the intensity of the large splitting was greatly enhanced. According to these results the two pairs of satellites do not stem from the presence of calcium ions and hydroxyapatite but originate from the presence of two groups of collagen fibers with different degrees of hydration. The enhanced intensity of the large splitting is attributed to an increased amount of water molecules that fill the void, resulting from the removal of hydroxyapatite, which resides near the fibers responsible for the large splitting. The quadrupolar splitting observed in the trabecular bone was not orientation-dependent, indicating a random orientation of the collagen fibers in that tissue.

The role of viscoelasticity of collagen fibers in articular cartilage: axial tension versus compression

The role of viscoelasticity of collagen fibers in bovine articular cartilage was examined in compression and tension using stress relaxation measurements in the axial direction (normal to the articular surface). Experimentally, for a given axial strain, both peak and equilibrium loads were higher in tension than in compression, whereas stress relaxation was stronger in compression, as indicated by the higher peak-to-equilibrium ratios. A viscoelastic fibril-reinforced model including fluid flow was used for analysis of the experimental data. The collagen fibrillar matrix was assumed to be viscoelastic with a strain-dependent tensile modulus, and the nonfibrillar matrix was modeled as linearly elastic. For axial tension, collagen viscoelasticity was found to account for most of the stress relaxation, while the effects of fluid pressurization on the tensile stress were negligible. In contrast, for axial compression, the dominant mechanism for stress relaxation arose from fluid pressurization, while the associated relaxation in collagen fibers mainly resulted in an increase in radial strain. The effective Poisson's ratio, defined as the ratio of the radial and axial strains, was generally smaller in compression than in tension, and deviated from the true Poisson's ratio in tensile tests because of the frictional contacts between the specimen and the loading platens. Furthermore, lower collagen elasticity in the axial direction was observed than in the radial direction. This study illustrates the essential role of collagen viscoelasticity and interstitial fluid pressurization in the mechanical response of articular cartilage.

Mapping the fiber orientation in articular cartilage at rest and under pressure studied by 2H double quantum filtered MRI.

The one-dimensional (2)H double quantum filtered (DQF) spectroscopic imaging technique was used to study the orientation of collagen fibers in articular cartilage. The method detects only water molecules in anisotropic environments, which in cartilage is caused by their interaction with the collagen fibers. A large quadrupolar splitting was observed in the calcified zone and a smaller splitting in the radial zone. In the transitional zone the splitting was not resolved and a small splitting was again detected in the superficial zone. From measurements performed at two orientations of the plug relative to the magnetic field it was deduced that in the calcified and radial zones the fibers are oriented perpendicular to the bone, bending at the transitional zone and flattening at the superficial zone. The effect of load applied to the cartilage-bone plug was monitored by the same technique. At low loads there is a small decrease in the quadrupolar splitting in the calcified zone, a marked decrease in the radial zone, and an increase of the splitting accompanied by a thickening of the superficial zone. Under high loads, while the thickening and the splitting of the superficial zone further increase, the splitting in the radial and calcified zones completely collapse. Pressure-induced changes in the thickness of the surface zone indicate flattening of the collagen fibers near the surface. The marked collapse of the splitting near the bone at high pressures may result from crimping of the collagen fibers.

Deformation of loaded articular cartilage prepared for scanning electron microscopy with rapid freezing and freeze-substitution fixation.

To investigate the effect of joint loading on collagen fibers in articular cartilage, 45 knees of adult rabbits were examined by scanning electron microscopy. The knees were loaded at the patella with a simulated "quadriceps force" of 0.5-4 times body weight for 0.5 or 25 minutes, plunge-frozen, and fixed by freeze-substitution with aldehydes. Six knees were loaded for 3 hours and then fixed conventionally. Fixed tibial plateaus were examined and then freeze-fractured through the area of tibiofemoral contact, dried, coated, and examined by scanning electron microscopy to assess the overall deformation of the tibial articular surface and matrix collagen fibers. With tissue prepared by conventional fixation used as a standard, the quality of fixation was graded by light and transmission electron microscopy of patellar cartilage taken from half of the freeze-fixed knees. In loaded specimens, an indentation was present where the femur contacted the tibial plateau. The diameter and apparent depth of the dent were proportional to the magnitude and duration of the load; no dent was seen in the controls. The thickness of the cartilage at the center of the indentation was reduced 15-80%. Meniscectomy always produced larger deformations in otherwise equivalent conditions. Icecrystal damage to cells was evident by transmission electron microscopy and scanning electron microscopy, but at magnifications as high as x 30,000 the collagen fibrils prepared by freeze-substitution and conventional aqueous methods were identical. In loaded regions, the collagen matrix of the tibial cartilage was deformed in two ways: (a) radial collagen fibers exhibited a periodic crimp, and (b) in regions where an indentation was created by the femoral condyle, the radial fibers were bent, in effect creating a tangential zone where none had existed before. The radial fibers apparently are loaded axially and buckle under normal loads.

The Collagenous Architecture of Articular Cartilage

The localization and directional orientation of collagen fibers in articular cartilage is demonstrated by scanning electron microscopy and polarized light microscopy. Vertical sections of articular cartilage show different directional orientations of collagen fibers through all zones of cartilage depending upon whether the sections are parallel or perpendicular to the cleft pattern produced when the surface of articular cartilage is pierced with a round pointed awl. Sections parallel to the cleft axis show a significant population of oblique collagen fibers which are not seen in sections perpendicular to the clefts. These oblique fiber groups show a progression from nearly radial to nearly tangential orientation from deep to more superficial zones, with the most abrupt directional change seen through the transitional zone. Within the transitional zone there is a narrow band having no vertical or horizontal collagen fibers and in which collagen fibers intersect predominantly at angles ranging between 45 and 135 degrees. The number of chondrocyte lacunae per unit area is greater in sections parallel to the cleft axis compared to perpendicular sections by a factor of approximately 1.6:1. There is therefore a greater relative number of chondrocytes in the plane of section having the greatest abundance of oblique collagen fibers, suggesting a cellular basis for the collagenous architecture observed. The results are consistent with published biophysical data relating tensile and swelling properties of all zones of articular cartilage to the cleft axis.