Differential Interference Contrast Optical Microscopy Research Articles

Thermo-Resistive Phase Behavior of Trivalent Ion-Induced Microscopic Protein-Rich Phases: Correlating with Ion-Specific Protein Hydration

Proteins, in the presence of trivalent cations, exhibit intriguing phase behavior which is contrasting compared to mono- and divalent cations. At room temperature (RT), trivalent cations induce microscopic liquid-liquid phase separation (LLPS) in which a protein-rich phase coexists with a dilute phase. The critical solution temperature related phenomena in these complex fluids are well studied; however, such studies have mostly been restricted below the denaturation temperature (TM) of the protein(s) involved. Here, we probe the phase behavior of bovine serum albumin (BSA) incubated at 70 °C (>TM) in the presence of Na+, Mg2+, La3+, Y3+, and Ho3+ ions. BSA in the presence of mono- and bivalent ions forms an intense gel phase at 70 °C; however, the trivalent salts offer remarkable thermal resistivity and retain the fluid LLPS phase. We determine the microscopic phase behavior using differential interference contrast optical microscopy, which shows that the LLPS droplet structures in the M3+ ion-containing protein solutions prevail upon heating, whereas Mg2+ forms composed cross-linking gelation upon thermal incubation. We probe the interior environment of the protein aggregates by ps-resolved fluorescence anisotropy measurements using 8-anilino-1-naphthalenesulfonic acid (ANS) as an extrinsic fluorophore. It reveals that while the LLPS phase retains the rotational time constants upon heating, in the case of gelation, the immediate environment of ANS gets significantly perturbed. We investigate the explicit protein hydration at RT as well as at T > TM using the ATR THz-FTIR (1.5-22.5 THz) spectroscopy technique and found that hydration shows strong ion specificity and correlates the phase transition behavior.

Detection of subtle cartilage and bone tissue degeneration in the equine joint using polarisation-sensitive optical coherence tomography

To explore the ability of polarisation-sensitive optical coherence tomography (PS-OCT) to rapidly identify subtle signs of tissue degeneration in the equine joint. Polarisation-sensitive optical coherence tomography (PS-OCT) images were systematically acquired in four locations along the medial and lateral condyles of the third metacarpal bone in five dissected equine specimens. Intensity and retardation PS-OCT images, and anomalies observed therein, were then compared and validated with high resolution images of the tissue sections obtained using Differential Interference contrast (DIC) optical light microscopy. The PS-OCT system was capable of imaging the entire equine osteochondral unit, and allowed delineation of the three structurally differentiated zones of the joint, that is, the articular cartilage matrix, zone of calcified cartilage and underlying subchondral bone. Importantly, PS-OCT imaging was able to detect underlying matrix and bone changes not visible without dissection and/or microscopy. PS-OCT has substantial potential to detect, non-invasively, sub-surface microstructural changes that are known to be associated with the early stages of joint tissue degeneration.

Subchondral bone plate thickness is associated with micromechanical and microstructural changes in the bovine patella osteochondral junction with different levels of cartilage degeneration

The influence of joint degeneration on the biomechanical properties of calcified cartilage and subchondral bone plate at the osteochondral junction is relatively unknown. Common experimental difficulties include accessibility to and visualization of the osteochondral junction, application of mechanical testing at the appropriate length scale, and availability of tissue that provides a consistent range of degenerative changes. This study addresses these challenges.A well-established bovine patella model of early joint degeneration was employed, in which micromechanical testing of fully hydrated osteochondral sections was carried out in conjunction with high-resolution imaging using differential interference contrast (DIC) optical light microscopy. A total of forty-two bovine patellae with different grades of tissue health ranging from healthy to mild, moderate, and severe cartilage degeneration, were selected. From the distal–lateral region of each patella, two adjacent osteochondral sections were obtained for the mechanical testing and the DIC imaging, respectively. Mechanical testing was carried out using a robotic micro-force acquisition system, applying compression tests over an array (area: 200 μm × 1000 μm, step size: 50 μm) across the osteochondral junction to obtain a stiffness map. Morphometric analysis was performed for the DIC images of fully hydrated cryo-sections. The levels of cartilage degeneration, DIC images, and the stiffness maps were used to associate the mechanical properties onto the specific tissue regions of cartilage, calcified cartilage, and subchondral bone plate.The results showed that there were up to 20% and 24% decreases (p < 0.05) in the stiffness of calcified cartilage and subchondral bone plate, respectively, in the severely degenerated group compared to the healthy group. Furthermore, there were increases (p < 0.05) in the number of tidemarks, bone spicules at the cement line, and the mean thickness of the subchondral bone plate with increasing levels of degeneration.The decreasing stiffness in the subchondral bone plate coupled with the presence of bone spicules may be indicative of a subchondral remodeling process involving new bone formation. Moreover, the mean thickness of the subchondral bone plate was found to be the strongest indicator of mechanical and associated structural changes in the osteochondral joint tissues.

Undulated Step Structure on the (0001¯) Facet of Physical Vapor Transport-Grown 4H-SiC Crystals.

The step structure on the (000)C facet of 4H-SiC boules grown by the physical vapor transport growth method with different nitrogen doping concentrations was examined in various scales, using different types of microscopy, such as differential interference contrast optical microscopy (DICM) and atomic force microscopy (AFM). DICM observations unveiled characteristic macroscopic surface features of the facet dependent on the nitrogen doping concentration. AFM observations revealed the existence of step trains of half unit-cell height (0.5 nm) on the facet and found that their separation was undulated with a characteristic wavelength dependent on the nitrogen doping concentration; the higher the nitrogen concentration, the longer was the undulation wavelength of step separation. Based on these results, we discussed the origin and formation mechanism of the separation-undulated step structure observed on the (000)C facet of nitrogen-doped 4H-SiC boules.

The micro and ultrastructural anatomy of bone spicules found in the osteochondral junction of bovine patellae with early joint degeneration.

The structural changes in the tissues of the osteochondral junction are a topic of interest, especially considering how bone changes are involved in the initiation and progression of osteoarthritis (OA). Our research group has previously demonstrated that at the cement line boundary between the zone of calcified cartilage (ZCC) and the subchondral bone, in mature bovine patellae with early OA, there are numerous bone spicules that have emerged from the underlying bone. These spicules contain a central vascular canal and a bone cuff. In this study, we use high-resolution differential interference contrast optical microscopy and scanning electron microscopy to compare the cartilage-bone junction of three groups of mature bovine patellae showing healthy to mild to moderately degenerate cartilage. The ZCC and bone junction was carefully examined to estimate the frequency of marrow spaces, bone spicules and fully formed bone bulges. The results reveal that bone spicules are associated with all grades of cartilage tissue studied, with the most occurring in the intermediate stages of tissue health. The micro and ultrastructure of the bone spicule are consistent with that of an osteon, especially those found in compression zones in long bones. Also considering the coexistence of marrow spaces and fully formed bone, this study suggests that these bone spicules arise similar to the formation of osteons in the bone remodelling process. The significance of this conclusion is in the way researchers approach the bone formation issue in the early degenerative joint. Instead of endochondral ossification, we propose that bone formation in OA is more akin to a combination of primary bone remodelling and de novo bone formation.

Macroscopically healthy articular cartilage with fibrillar-scale early tissue degeneration subject to impact loading results in greater extent of cell-death

From previous investigations it has been shown that there exists healthy-appearing articular cartilage that contains collagen fibril network destructuring. It is hypothesised that such sub-micron scale destructuring not only presents an increased vulnerability to tissue scale damage following impact loading, but an increase in cell death as well.Cartilage-on-bone blocks from 12 patellae, six healthy (G0) and the other six with sub-micron fibrillar destructuring (G1), were obtained and subject to 2.3 J impact loading. Two sets of sub-samples were obtained for each block tested. One set was used to examine for the live/dead cell response using calcein-AM and propidium iodide staining, imaged with confocal microscopy. The tissue microstructural matrix was imaged from the other matched set, unstained and in its fully hydrated state, using differential interference contrast optical light microscopy. High speed imaging of the impact was used to calculate the velocity changes or coefficient of restitution (COR) and used as a proxy of energy that the tissue absorbed. A previously defined tissue matrix damage score was used to quantify the extent of fracturing and cracking in the matrix. The cell death (PCD) was counted and presented as a percentage against all cells live plus dead.The energy absorbed was 36.5% higher in G1 than in G0 (p = 0.034). However, the damage score and PCD of samples in the G1 group was much larger than the G0 group, ~300% and 161% respectively. Microscopy showed that cell death is associated to both matrix compaction and further fibrillar destructuring from the ECM to the territorial matrix regions of the chondron.Following impact loading, cartilage tissue that appears normal but contains sub-micron fibrillar matrix destructuring responds with significantly increased cell death.

Protein Levels and Microstructural Changes in Localized Regions of Early Cartilage Degeneration Compared with Adjacent Intact Cartilage.

It was hypothesized that the respective protein profiles of bovine cartilage from sites of localized mild to moderate (GI to GII) degeneration versus adjacent sites of intact tissue would vary in accordance with the tissue microstructural changes associated with a pre-osteoarthritic state. A total of 15 bovine patellae were obtained for this study. Paired samples of tissue were collected from the lateral region of each patella. If the patella contained a site of degeneration, a paired tissue set involved taking one sample each from the degenerated site and the intact tissue adjacent to it. Sufficient tissue was collected to facilitate 2 arms of investigation: microstructural imaging and proteome analysis. The microstructural analysis used a bespoke tissue preparation technique imaged with differential interference contrast optical microscopy to assess fibrillar scale destructuring and underlying bone spicule formation. An iTRAQ-based proteome analysis was performed using liquid chromatography-tandem mass spectrometry to identify the differential levels of proteins across the intact and degenerated cartilage and further, the results were validated with multiple reaction monitoring assay. In the healthy cartilage pairs, there was no significant variation in protein profiles between 2 adjacent sample sites. In pairs of tissue that contained a sample of GI/GII tissue, there were both significant microstructural changes as well as the difference in abundance levels of 24 proteins. From the known functions of the 24 proteins, found to be strongly aligned with the specific microstructural changes observed, a unique "proteins ensemble" involved in the initiation and progression of early cartilage degeneration is proposed.

A Microstructural Investigation of Disc Disruption Induced by Low Frequency Cyclic Loading.

Microstructural investigation of low frequency cyclic loading and flexing of the lumbar disc. To explore micro-level structural damage in motion segments subjected to low frequency repetitive loading and flexing at sub-acute loads. Cumulative exposure to mechanical load has been implicated in low back pain and injury. The mechanical pathways by which cyclic loading physically affects spine tissues remain unclear, in part due to the absence of high quality microstructural evidence. The study utilized seven intact ovine lumbar spines and from each spine one motion segment was used as a control, two others were cyclically loaded. Ten motion segments were subjected to 5000 cycles at 0.5 Hz with a peak load corresponding to ∼30% of that required to achieve failure. An additional small group of segments subjected to 10,000 or 30,000 cycles was similarly analyzed. Following chemical fixation and decalcification samples were cryosectioned along one of the oblique fiber angles and imaged in their fully hydrated state using differential interference contrast optical microscopy. Structural damage obtained from the images was organized into an algebraic shell for analysis. At 5000 cycles the disc damage was limited to inner wall distortions, evidence of stress concentrations at bridging-lamellae attachments, and small delaminations. The high-cycle discs tested exhibited significant mid-wall damage. There was no evidence of nuclear material being displaced. At this low frequency and without the application of sustained loading or a more severe loading regime, or maintaining a constant flexion with repetitive loading, it seems unlikely that actual nuclear migration occurs. It is possible that the inner-annular damage shown in the low dose group could disrupt pathways for nutrient diffusion leading to earlier cell death and matrix degradation, thus contributing to a cascade of degeneration. N/A.

The influence of early degenerative changes on the vulnerability of articular cartilage to impact-induced injury

BackgroundRecently, the structural changes in a bovine model of early degeneration were validated by our research group to be analogous to that in early human osteoarthritis. The hypothesis of this study was that the structural changes associated with increasing levels of degeneration would lead to higher levels of tissue damage in response to impact induced injury. MethodsA total of forty bovine patellae were obtained for this study. Cartilage-on-bone samples were extracted from the distal lateral quarter, a region known to be affected by varying levels of degeneration. A single impact drop test was applied to these samples delivering 2.3J of energy. A dynamic load cell and image capture at 2000fps allowed for the calculation of the reaction stress and coefficient of restitution. The extent of tissue damage was examined from the micro to ultrastructural levels using differential interference contrast optical microscopy and scanning electron microscopy respectively. FindingsThe impact mechanical properties of mildly degenerate articular cartilage were not significantly different but showed a significantly larger amount of structural damage. From comparing the mechanical and structural response of intact and mildly degenerate cartilage, to tissue showing increased macro-scale tissue degeneration, the significance of the surface layer and fibrillar scale transverse interconnectivity in effectively attenuating impact loads is demonstrated in this study. InterpretationThis study shows that even though articular cartilage can appear visibly normal under macroscopic observation, the micro-scale structural changes associated with very early stage osteoarthritis can have a significant effect on its vulnerability to impact damage.

How maturity influences annulus-endplate integration in the ovine intervertebral disc: a micro- and ultra-structural study.

The annulus-endplate anchorage system plays a vital role in structurally linking the compliant disc to its adjacent much more rigid vertebrae. Past literature has identified the endplate as a region of weakness, not just in the mature spine but also in the immature spine. The aim of this structural study was to investigate in detail the morphological changes associated with annulus-endplate integration through different stages of maturity. Ovine lumbar motion segments were collected from two immature age groups: (i) newborn and (ii) spring lamb (roughly 3months old); these were compared with a third group of previously analysed mature ewe samples (3-5years). Sections from the posterior region of each motion segment were obtained for microstructural analysis and imaged in their fully hydrated state via differential interference contrast (DIC) optical microscopy. Selected slices were further prepared and imaged via scanning electron microscopy (SEM) to analyse fibril-level modes of integration. Despite significant changes in endplate morphology, the annular fibre bundles in all three age groups displayed a similar branching mechanism, with the main bundle splitting into several sub-bundles on entering the cartilaginous endplate. This morphology, previously described in the mature ovine disc, is thought to strengthen significantly annulus-endplate integration. Its prevalence from an age as young as birth emphasizes the critical role that it plays in the anchorage system. The structure of the branched sub-bundles and their integration with the surrounding matrix were found to vary with age due to changes in the cartilaginous and vertebral components of the endplate. Microscopically, the sub-bundles in both immature age groups appeared to fade into the surrounding tissue due to their fibril-level integration with the cartilaginous endplate tissue, this mechanism being particularly complex in the spring lamb disc. However, in the fully mature disc, the sub-bundles remained as separate entities throughout the full depth of their anchorage into the cartilaginous endplate. Cell morphology was also found to vary with maturity within the cartilaginous matrix and it is proposed that this relates to endplate development and ossification.

Surface morphology and step instability on the (0001̄)C facet of physical vapor transport-grown 4H–SiC single crystal boules

Surface morphologies on the (0001¯) C facet of 4H–SiC boules grown using the physical vapor transport method were examined in various scales (from millimeter to nanometer) using different types of microscopies such as differential interference contrast (DIC) optical microscopy and atomic force microscopy (AFM). DIC optical microscopic observation revealed that there are three distinct morphological regions at the growth front of the 4H–SiC boules: the facetted region, non-facetted region and intermediate region between them. The local inclination of the facet surface from the {0001} basal plane increases toward the edge of the facetted region and then decreases in the intermediate region. AFM observations revealed that characteristic step structures were established in these two regions and that nitrogen doping significantly influenced the stability of the step structures. Based on the results, the formation mechanism of surface morphologies on the (0001¯) C facet of 4H–SiC boules is discussed.

A quantitative characterization of micro-gravity and unit-gravity processed ZBLAN glass

Micro-gravity processing has been proven to suppress crystallization in ZrF4–BaF2–LaF3–AlF3–NaF (ZBLAN) glass. Crystallization suppression and characterization is the next step towards the development of low-loss mid-Infrared (IR) fiber optics. This study focuses on the characterization of micro-gravity versus unit-gravity processed ZBLAN glass. A unique quantitative crystallization characterization technique was employed on the samples that utilize a combination of Differential Interference Contrast (DIC) optical microscopy with on board microscope counting and measuring software techniques. To date, past researchers who have studied the crystallization suppression of ZBLAN glass in micro-gravity have only characterized their material through visual inspection of micrographs. This study makes use of optical micrographs while providing additional quantitative data that has been lacking from previous studies. This novel characterization technique provides additional insight into understanding the crystallization behavior of ZBLAN glass that can lead to the development of low-loss mid IR fiber optics. The results from this study show that the quantification data agrees with the micrographs, however, the new results show a more precise degree of crystallization suppression through quantified data, such as crystallite size and amount per sample.

Observation of the Surface Morphology on the (0001)C Facet of 4H-SiC Boules

The surface morphology on the (000-1)C facet of 4H-SiC boules grown by the physical vapor transport method was examined in various scales (from millimeter to nanometer) using different types of microscopies such as differential interference contrast (DIC) optical microscopy and atomic force microscopy (AFM). The DIC optical microscope observation revealed that there exist three distinct morphological regions at the growth front of the 4H-SiC boules; they are facetted, non-facetted, and the intermediate region between them. The local inclination of the facet surface from the (000-1) basal plane increases toward the facet edge and then decreases over the intermediate region. AFM observations revealed characteristic step structures in these two regions and also that they are significantly influenced by nitrogen-doping. Based on the results, the formation mechanism of the facet morphology on 4H-SiC boules is discussed.

A multiscale structural investigation of the annulus-endplate anchorage system and its mechanisms of failure

Background contextThe annulus-endplate anchorage system performs a critical role in the disc, creating a strong structural link between the compliant annulus and the rigid vertebrae. Endplate failure is thought to be associated with disc herniation, a recent study indicating that this failure mode occurs more frequently than annular rupture. PurposeThe aim was to investigate the structural principles governing annulus-endplate anchorage and the basis of its strength and mechanisms of failure. Study designLoading experiments were performed on ovine lumbar motion segments designed to induce annulus-endplate failure, followed by macro- to micro- to fibril-level structural analyses. MethodsThe study was funded by a doctoral scholarship from our institution. Samples were loaded to failure in three modes: torsion using intact motion segments, in-plane tension of the anterior annulus-endplate along one of the oblique fiber angles, and axial tension of the anterior annulus-endplate. The anterior region was chosen for its ease of access. Decalcification was used to investigate the mechanical influence of the mineralized component. Structural analysis was conducted on both the intact and failed samples using differential interference contrast optical microscopy and scanning electron microscopy. ResultsTwo main modes of anchorage failure were observed—failure at the tidemark or at the cement line. Samples subjected to axial tension contained more tidemark failures compared with those subjected to torsion and in-plane tension. Samples decalcified before testing frequently contained damage at the cement line, this being more extensive than in fresh samples. Analysis of the intact samples at their anchorage sites revealed that annular subbundle fibrils penetrate beyond the cement line to a limited depth and appear to merge with those in the vertebral and cartilaginous endplates. ConclusionsAnnulus-endplate anchorage is more vulnerable to failure in axial tension compared with both torsion and in-plane tension and is probably due to acute fiber bending at the soft-hard interface of the tidemark. This finding is consistent with evidence showing that flexion, which induces a similar pattern of axial tension, increases the risk of herniation involving endplate failure. The study also highlights the important strengthening role of calcification at this junction and provides new evidence of a fibril-based form of structural integration across the cement line.

Characterization of scraper-shaped defects on 4H-SiC epitaxial film surfaces

We have found undiscovered defects on a 4H-SiC epitaxial layer, the shape of which resembles a scraper in images taken by confocal differential interference contrast optical microscopy. The surface morphological structure and formation mechanism of the scraper-shaped defects were investigated by atomic force microscopy and grazing incidence monochromatic synchrotron X-ray topography, respectively. The scraper-shaped defects were surface morphological defects consisting of surface asperity and were caused by the migration of interfacial dislocations. The evaluation of the thermal oxide reliability of metal–oxide–semiconductor capacitors fabricated on these defects was performed by time-dependent dielectric breakdown measurement. The degradation of thermal oxide occurred only on the downstream line of the scraper-shaped defects. The thickness of the oxide layer on these defects was also investigated using cross-sectional transmission electron microscopy.

A multi-scale structural study of the porcine anterior cruciate ligament tibial enthesis.

Like the human anterior cruciate ligament (ACL), the porcine ACL also has a double bundle structure and several biomechanical studies using this model have been carried out to show the differential effect of these two bundles on macro-level knee joint function. It is hypothesised that if the different bundles of the porcine ACL are mechanically distinct in function, then a multi-scale anatomical characterisation of their individual enthesis will also reveal significant differences in structure between the bundles. Twenty-two porcine knee joints were cleared of their musculature to expose the intact ACL following which ligament-bone samples were obtained. The samples were fixed in formalin followed by decalcification with formic acid. Thin sections containing the ligament insertion into the tibia were then obtained by cryosectioning and analysed using differential interference contrast (DIC) optical microscopy and scanning electron microscopy (SEM). At the micro-level, the anteromedial (AM) bundle insertion at the tibia displayed a significant deep-rooted interdigitation into bone, while for the posterolateral (PL) bundle the fibre insertions were less distributed and more focal. Three sub-types of enthesis were identified in the ACL and related to (i) bundle type, (ii) positional aspect within the insertion, and (iii) specific bundle function. At the nano-level the fibrils of the AM bundle were significantly larger than those in the PL bundle. The modes by which the AM and PL fibrils merged with the bone matrix fibrils were significantly different. A biomechanical interpretation of the data suggests that the porcine ACL enthesis is a specialized, functionally graded structural continuum, adapted at the micro-to-nano scales to serve joint function at the macro level.

Different microstructures of mobile twin boundaries in 10 M modulated Ni–Mn–Ga martensite

The morphology and microstructure of a single, macroscopically straight twin interface with a twinning stress of about 1MPa was analysed in detail by differential interference contrast optical microscopy and X-ray diffraction. The interface was identified as a Type I macrotwin boundary between two variants with a/b-laminates and constant modulation direction, in contrast with a highly mobile twinned interface consisting of Type II macrotwin boundary segments with changing modulation direction and a/b-laminate reported earlier. Theoretical analysis using elastic continuum theory shows that only pure Type I or Type II boundaries are fully compatible with a/b-laminate. Other hypothetical twin microstructures combining these two mobile interfaces are shown to be incompatible to various degrees. A weakly incompatible combination of Type I and II boundaries was experimentally observed. The large difference in mobility between Type I and Type II macrotwin boundaries created at the same location of the same sample indicates that the mobility depends on the internal structure of these boundaries. A possible origin of this different mobility is discussed.

Plasma-Enhanced Chemical Vapor Deposition of n-Heptane and Methyl Methacrylate for Potential Cell Alignment Applications

Plasma-enhanced chemical vapor deposited polymers (plasma polymers) are promising candidates for biomaterials applications. In the present study, plasma deposition as a fast and easily scalable method was adapted to deposit coatings from n-heptane and methyl methacrylate monomers onto glass substrates. Linear patterns with line and groove widths between 1.25 and 160 μm were introduced by degrative UV-lithography for cell alignment. Differential interference contrast optical microscopy, profilometry and atomic force microscopy revealed that the patterned surfaces had a smooth, homogeneous appearance and a pattern height of 8 and 45 nm for plasma deposited n-heptane and methyl methacrylate, respectively. UV-lithography increased the oxygen content on the surface drastically as shown by X-ray photoelectron spectroscopy. After immersion in simulated body fluid for 21 days, the pattern was still intact, and the ester groups were also maintained for the most part as shown by infrared spectroscopy. To test the coatings' potential applicability for biomaterial surfaces in a preliminary experiment, we cultured murine preosteoblastic MC3T3-E1 cells on these coatings. Light and electron microscopically, a normal spindle-shaped and aligned cell morphology was observed. At the mRNA level, cells showed no signs of diminished proliferation or elevated expression of apoptosis markers. In conclusion, plasma-enhanced chemical vapor deposited polymers can be patterned with a fast and feasible method and might be suitable materials to guide cell alignment.

Transmission Welding of Glass by Femtosecond Laser: Mechanism and Fracture Strength

Femtosecond laser pulses were focused on the interface of two glass specimens. Proper use of optical and laser processing parameters enables transmission welding. The morphology of the weld cross section was studied using differential interference contrast optical microscopy. In addition, a numerical model was developed to predict the absorption volumes of femtosecond laser pulses inside a transparent material. The model takes into account the temporal and spatial characteristics and propagation properties of the laser beam, and the transmission welding widths were subsequently compared with the absorption widths predicted by the model. The model can lead to the achievement of a desirable weld shape through understanding the effects of laser pulse energy and numerical aperture on the shape of the absorption volume. The changes in mechanical properties of the weld seams were studied through spatially resolved nanoindentation, and indentation fracture analysis was used to investigate the strength of the weld seams.

Macro-, micro- and ultrastructural investigation of how degeneration influences the response of cartilage to loading

Articular cartilage functions as a load-bearing tissue by virtue of a functional coupling between its hydrated proteoglycan component and its zonally differentiated fibrillar network. How degeneration influences this relationship at the macro-, micro-, and ultrastructural levels is investigated in this study. Healthy bovine patellae ( N = 9 ) and patellae exhibiting varying degrees of degeneration ( N = 16 ) formed the basis of the study. Cartilage-on-bone blocks obtained from each patella were subjected to creep loading under a nominal stress of 4.5 MPa via a rectangular planar indenter which incorporated a narrow channel relief space to create a defined region where the cartilage would not be directly loaded. Following the attainment of creep equilibrium each sample was chemically fixed while under load so as to preserve the deformed state of the cartilage matrix. The structural response of the matrix was then analysed using differential interference contrast (DIC) optical microscopy and scanning electron microscopy (SEM). The morphology of the cartilage matrix extruded into the channel relief region was dramatically influenced by the severity of degeneration. The microscopic and ultrastructural characteristics of the extruded matrix showed that the load response of bulk cartilage is determined primarily by the microstructural integrity of the strain-limiting tangential layer and the nano-level interconnectivity of the fibrillar network. In conclusion, this study showed that three mechanically significant structural features of cartilage are important: (1) the strain limiting surface layer; (2) the micro-level boundaries in its zonally differentiated structure, and (3) the extent of fibrillar interconnectivity. Degeneration degrades or destroys the articular surface and ‘destructures’ the fibrillar network such that the latter functions less effectively as a proteoglycan entrapment system.