The ultrastructure of cartilage tissue and its swelling response in relation to matrix health.

This multi-length scale anatomical study explores the influence of mild cartilage structural degeneration on the tissue swelling response. While the swelling response of cartilage has been studied extensively, this is the first study to reveal and correlate tissue microstructure and ultrastructure, with the swelling induced cartilage tissue strains. Cartilage sample strips (n = 30) were obtained from the distal-lateral quadrant of thirty mildly degenerate bovine patellae and, following excision from the bone, the cartilage strips were allowed to swell freely for 2 h in solutions of physiological saline and distilled water successively. The swelling response of this group of samples were compared with that of healthy cartilage, with (n = 20) and without the surface layer (n = 20). The subsequent curling response of cartilage showed that in healthy tissue it was highly variable, and with the surface removed some samples curved in the opposite direction, while in the mildly degenerate tissue group, virtually all tissue strips curved in a consistent upward manner. A significant difference in strain was observed between healthy samples with surface layer removed and mildly degenerate samples, illustrating how excision of the surface zone from pristine cartilage is insufficient to model the swelling response of tissue which has undergone natural degenerative changes. On average, total tissue thickness increased from 940 µm (healthy) to 1079 µm (mildly degenerate), however, looking at the zonal strata, surface and transition zone thicknesses both decreased while deep zone thickness increased from healthy to mildly degenerate tissue. Morphologically, changes to the surface zone integrity were correlated with a diminished surface layer which, at the ultrastructural scale, correlated with a decreased fibrillar density. Similarly, fibrosity of the general matrix visible at the microscale was associated with a loss of later interconnectivity resulting in large, aggregated fibril bundles. The microstructural and ultrastructural investigation revealed that the key differences influencing the tissue swelling strain response was (1) the thickness and extent of disruption to the surface layer and (2) the amount of fibrillar network destructuring, highlighting the importance of the collagen and tissue matrix structure in restraining cartilage swelling.

Age-related differences in hamstring tendon used as autograft in reconstructive anterior cruciate ligament surgery.

PURPOSE: The hamstring tendon is the most commonly used autograft material in reconstructive surgeries of anterior cruciate ligament (ACL) tears. Younger patients have worse surgical outcomes, with a higher risk of re-rupture. We hypothesized that age-related changes in hamstring tendon properties affect the tendon's propensity to rupture when used as an autograft in ACL reconstructions. The purpose of this study was to compare hamstring tendon samples obtained from people aged 20 years or younger to samples obtained from older people. METHODS: Superfluous hamstring tendon material was collected from 13 young donors (aged 16-20 years) and 17 older donors undergoing ACL reconstructive surgery. Sections of the tendon samples were used for biomechanical testing, structural analysis of collagen fibrils by electron microscopy, and global analysis of gene expression by microarrays. RESULTS: We found that tendon samples from the older group had lower Young's modulus than the younger group (P = 0.015), whereas the stress to failure was similar in the two groups. We found no difference in the average diameter of collagen fibrils between the two groups. Microarray analysis identified 162 differentially expressed genes (fold change ≥ 1.5, P < 0.05), with overrepresentation of several biological processes, including regulation of adhesion, migration, inflammation, and differentiation (fold enrichment > 2.0, false discovery rate P < 0.05). CONCLUSION: The hamstring tendon from younger people has higher stiffness than tendon from older people, and the profile of gene expression in tendon varies with age. These differences may negatively affect the performance of the hamstring tendon in ACL reconstructions in younger people.

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.

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 3 months old); these were compared with a third group of previously analysed mature ewe samples (3-5 years). 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.

A more realistic disc herniation model incorporating compression, flexion and facet-constrained shear: a mechanical and microstructural analysis. Part I: Low rate loading.

PURPOSE: To date, the mechanisms of disc failure have been explored at a microstructural level in relatively simple postures. However, in vivo the disc is known to be subjected to complex loading in compression, bending and shear, and the influence of these factors on the mechanisms of disc failure is yet to be described at a microstructural level. The purpose of this study was to provide a microstructural analysis of the mechanisms of failure in healthy discs subjected to compression while held in a complex posture incorporating physiological amounts of flexion and facet-constrained shear. METHODS: 30 motion segments from 10 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object, and at a displacement rate of 40 mm/min. Nine of the 30 samples reached the predetermined displacement prior to a reduction in load and were classified as early-stage failures, providing insight into initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy. RESULTS: Complex postures significantly reduced the load required to cause disc failure than earlier described for flexed postures [8.42 kN (STD 1.22 kN) compared to 9.69 kN (STD 2.56 kN)] and resulted in a very different failure morphology to that observed in either simple flexion or direct compression, involving infiltration of nucleus material in a circuitous path to the annular periphery. CONCLUSION: The complex posture as used in this study significantly reduced the load required to cause disc failure, providing further evidence that asymmetric postures while lifting should be avoided if possible.

A more realistic disc herniation model incorporating compression, flexion and facet-constrained shear: a mechanical and microstructural analysis. Part II: high rate or 'surprise' loading.

PURPOSE: Part I of this study explored mechanisms of disc failure in a complex posture incorporating physiological amounts of flexion and shear at a loading rate considerably lower than likely to occur in a typical in vivo manual handling situation. Given the strain-rate-dependent mechanical properties of the heavily hydrated disc, loading rate will likely influence the mechanisms of disc failure. Part II investigates the mechanisms of failure in healthy discs subjected to surprise-rate compression while held in the same complex posture. METHODS: 37 motion segments from 13 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object at a displacement rate of 400 mm/min. Seven of the 37 samples reached the predetermined displacement prior to a reduction in load and were classified as early stage failures, providing insight to initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy. RESULTS: The average failure load under high rate complex loading was 6.96 kN (STD 1.48 kN), significantly lower statistically than for low rate complex loading [8.42 kN (STD 1.22 kN)]. Also, unlike simple flexion or low rate complex loading, direct radial ruptures and non-continuous mid-wall tearing in the posterior and posterolateral regions were commonly accompanied by disruption extending to the lateral and anterior disc. CONCLUSION: This study has again shown that multiple modes of damage are common when compressing a segment in a complex posture, and the load bearing ability, already less than in a neutral or flexed posture, is further compromised with high rate complex loading.

Crimp morphology in the ovine anterior cruciate ligament.

While the crimp morphology in ligaments and tendons has been described in detail in the literature, its relative distribution within the tissue has not been studied, especially in relation to the complex multi-bundle arrangement as is found in the anterior cruciate ligament (ACL). In this study, the crimp morphology of the ovine ACL was examined topologically and with respect to its double-bundle structure. The crimp morphologies were compared with the knee in three knee positions, namely stance, maximum extension and maximum flexion. As a control, the crimp morphology of the ACL free from its bony attachments was determined. In the control samples, the anterior-medial (AM) bundle contained a combination of coarse and fine crimp, whereas the posterior-lateral (PL) bundle manifested only a coarse crimp. Using the extent of crimp loss observed when subjecting the knee to the respective positions, and comparing with the controls, the crimp morphologies show that the AM bundle of the ACL is most active in the stance position, whereas for the maximum extension and flexion positions the PL bundle is most active. We propose that these differences in crimp morphologies have relevance to ACL design and function.

How a radial focal incision influences the internal shear distribution in articular cartilage with respect to its zonally differentiated microanatomy.

Articular surface fibrillation and the loss of both transverse interconnectivity and zonal differentiation are indicators of articular cartilage (AC) degeneration. However, exactly how these structural features affect the load-redistributing properties of cartilage is still poorly understood. This study investigated how a single radial incision made to varying depths with respect to the primary zones of AC influenced its deformation response to compression. Three depths of incision were applied to cartilage-on-bone tissue blocks: one not exceeding the transition zone; one into the mid-radial zone; and one down to the calcified cartilage. Also included were non-incised controls. All samples were compressed to a near-equilibrium strain using a flat-faced indenter that incorporated a central relief channel within which the incision could be positioned lengthwise along the channel axis. Employing fixation under load followed by decalcification, the structural responses of the cartilage-on-bone samples were investigated. The study provides an analysis of the micro-morphological response that is characteristic of a completely normal cartilage-on-bone system but which contains a defined degree of disruption induced by the focal radial incision. The resulting loss of transverse continuity of the cartilage with respect to its zonally differentiated structure is shown to lead to an altered pattern of internal matrix shear whose intensity varies with incision depth.

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.

Microstructural changes in cartilage and bone related to repetitive overloading in an equine athlete model.

The palmar aspect of the third metacarpal (MC3) condyle of equine athletes is known to be subjected to repetitive overloading that can lead to the accumulation of joint tissue damage, degeneration, and stress fractures, some of which result in catastrophic failure. However, there is still a need to understand at a detailed microstructural level how this damage progresses in the context of the wider joint tissue complex, i.e. the articular surface, the hyaline and calcified cartilage, and the subchondral bone. MC3 bones from non-fractured joints were obtained from the right forelimbs of 16 Thoroughbred racehorses varying in age between 3 and 8 years, with documented histories of active race training. Detailed microstructural analysis of two clinically important sites, the parasagittal grooves and the mid-condylar regions, identified extensive levels of microdamage in the calcified cartilage and subchondral bone concealed beneath outwardly intact hyaline cartilage. The study shows a progression in microdamage severity, commencing with mild hard-tissue microcracking in younger animals and escalating to severe subchondral bone collapse and lesion formation in the hyaline cartilage with increasing age and thus athletic activity. The presence of a clearly distinguishable fibrous tissue layer at the articular surface immediately above sites of severe subchondral collapse suggested a limited reparative response in the hyaline cartilage.

Squatting-related tibiofemoral shear reaction forces and a biomechanical rationale for femoral component loosening.

Previous gait studies on squatting have described a rapid reversal in the direction of the tibiofemoral joint shear reaction force when going into a full weight-bearing deep knee flexion squat. The effects of such a shear reversal have not been considered with regard to the loading demand on knee implants in patients whose activities of daily living require frequent squatting. In this paper, the shear reversal effect is discussed and simulated in a finite element knee implant-bone model, to evaluate the possible biomechanical significance of this effect on femoral component loosening of high flexion implants as reported in the literature. The analysis shows that one of the effects of the shear reversal was a switch between large compressive and large tensile principal strains, from knee extension to flexion, respectively, in the region of the anterior flange of the femoral component. Together with the known material limits of cement and bone, this large mismatch in strains as a function of knee position provides new insight into how and why knee implants may fail in patients who perform frequent squatting.

Limited forearm motion compensated by thoracohumeral kinematics when performing tasks requiring pronation and supination.

This study investigates the altered thoracohumeral kinematics when forearm rotation is restricted while performing five activities requiring pronation and supination. Two splints simulated both a fixed-supinated or fixed-neutral forearm in six healthy subjects; the three-dimensional coupled relationship among motion about the forearm, elbow, and shoulder were analyzed. In using a screwdriver, the normal range of forearm rotation of 77.6° (SD = 30.8°) was reduced in the fixed-supinated to 11.3° (SD = 2.9°) and fixed-neutral to 18.2° (SD = 6.2°). This restriction from the fixed-supinated and fixed-neutral forearms was compensated at the shoulder by a significant increase in the total range of (1) ad/abduction by 57.3° and 62.8° respectively (p < .001), (2) forward-reverse flexion (24.3° and 18.2° respectively; p < .05) and (3) internal-external rotation (37.1° and 44.2° respectively; p < .001). A similar result was demonstrated for the doorknob activity. The elbow did not significantly contribute to forearm rotation (p = .14), and is believed to be due to the elbow axis being orthogonal and oblique to the forearm axis. For open kinetic-chain activities, with a fixed-supinated forearm performing there was a significant coupled increase in ad/abduction (p < .05) and int/external rotation (p < .05) for the phone and feeding tasks, with the phone task also having a significantly increased forward shoulder flexion (p < .05). For the fixed-neutral forearm, significant compensatory movement was only seen in the feeding task with increased ad/abduction and internal-external shoulder rotation (p < .05) and the card inserting task with increased ad/abduction and forward-reverse shoulder flexion. Limited forearm function requires compensatory motion from adjacent joints to perform activities that require pronation and supination.

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.

Impact-induced cartilage damage assessed using polarisation-sensitive optical coherence tomography.

Non-invasive determination of structural changes in articular cartilage immediately after impact and rehydration provides insight into the response and recovery of the soft tissue, as well as provides a potential methodology for clinicians to quantify early degenerative changes. In this study, we use polarisation-sensitive optical coherence tomography (PS-OCT) to examine subtle alterations of the optical properties in healthy and early-stage degenerate articular cartilage immediately after impact loading to identify structurally relevant metrics required for understanding the mechanical factors of osteoarthritic initiation and progression. A custom-designed impact testing rig was used to deliver 0.9 J and 1.4 J impact energies to bovine articular cartilage. A total of 52 (n=26 healthy, n=26 mildly degenerate) cartilage-on-bone samples were imaged before, immediately after, and 3 h after impact. PS-OCT images were analyzed to assess changes relating to surface irregularity, optical attenuation, and birefringence. Mildly degenerate cartilage exhibits a significant change in birefringence following 1.4 J impact energies compared to healthy samples which is believed to be attributable to degenerate cartilage being unable to fully utilise the fluid phase to distribute and dampen the energy. After rehydration, the polarisation-sensitive images appear to 'optically-recover' reducing the reliability of birefringence as an absolute metric. Surface irregularity and optical attenuation encode diagnostically relevant information and may serve as markers to predict the mechanical response of articular cartilage. PS-OCT with its ability to non-invasively image the sub-surface microstructural abnormalities of cartilage presents as an ideal modality for cartilage degeneration assessment and identification of mechanically vulnerable tissue.

The dynamic impact behavior of the human neurocranium.

Realistic biomechanical models of the human head should accurately reflect the mechanical properties of all neurocranial bones. Previous studies predominantly focused on static testing setups, males, restricted age ranges and scarcely investigated the temporal area. This given study determined the biomechanical properties of 64 human neurocranial samples (age range of 3 weeks to 94 years) using testing velocities of 2.5, 3.0 and 3.5 m/s in a three-point bending setup. Maximum forces were higher with increasing testing velocities (p ≤ 0.031) but bending strengths only revealed insignificant increases (p ≥ 0.052). The maximum force positively correlated with the sample thickness (p ≤ 0.012 at 2.0 m/s and 3.0 m/s) and bending strength negatively correlated with both age (p ≤ 0.041) and sample thickness (p ≤ 0.036). All parameters were independent of sex (p ≥ 0.120) apart from a higher bending strength of females (p = 0.040) for the 3.5 -m/s group. All parameters were independent of the post mortem interval (p ≥ 0.061). This study provides novel insights into the dynamic mechanical properties of distinct neurocranial bones over an age range spanning almost one century. It is concluded that the former are age-, site- and thickness-dependent, whereas sex dependence needs further investigation.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Articular cartilage compression: how microstructural response influences pore pressure in relation to matrix health.

Our research investigated the influence of degeneration on both the pore-pressure development and microstructural response of cartilage during indentation with a flat-porous-indenter. Experiments were conducted to link the mechanical and structural responses of normal and degenerate articular cartilage. We found that from the instant of loading the degenerate matrix generated a higher peak hydrostatic excess pore pressure in a shorter period of time than the normal matrix. Following the attainment of this peak value the pore pressure in both tissue groups then gradually decayed toward zero over time, thus demonstrating a classical consolidation response. The microstructural analysis provided a unique insight into the influence of degeneration on the mechanisms of internal stress-sharing within the loaded matrix. Both disruption of the articular surface and general matrix destructuring results in an altered deformation field in both the directly loaded and nondirectly loaded regions. It is argued that the higher levels of matrix shear combined with less of the applied load being redirected into the wider cartilage continuum accounts for the elevated levels of peak hydrostatic pore pressure generated in the degenerate matrix.

Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

OBJECTIVE: The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. DESIGN: Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. RESULTS: Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. CONCLUSION: This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

The mechanical significance of the zonally differentiated collagen network of articular cartilage in relation to tissue swelling.

BACKGROUND: We hypothesise that the Benninghoff arcade fibril structure motif of cartilage is able to predict the swelling response of cartilage. METHODS: A total of ten healthy adult bovine patellae were used for this study, yielding 20 paired full depth cartilage samples (half with surface layer intact and half with surface layer removed). Following excision from the bone, samples were allowed to equilibrate first in physiological saline for 2 h, and then in distilled water for another 2 h to maximise the swelling response. Images were captured using a stereomicroscope to measure strain and the fully-swollen samples were fixed in 10% formalin to retain shape for microscopic and ultrastructural imaging. FINDINGS: We expected all swelling samples with an intact 'strain-limiting' surface layer to curl upwards, instead only 70% of them did. For samples without a surface layer, we expected the swelling to be evenly distributed and to remain relatively uncurled; but in 40% of the samples there was a downward curvature (i.e. opposite to that of the previous group). Micro-to-ultrastructural imaging, to determine fibrillar structure and organisation, revealed the deep zone cartilage was an additional counter layer limiting swelling strain, and was the likely cause of the unexpected swelling responses. INTERPRETATION: Our expectations that the surface layer alone will influence the swelling response, was based on the assumptions of the Benninghoff arcade model. This study highlights the additional importance of sub-micron scale fibrillar interconnectivity and the role of the deep zone.

Raman microspectroscopic analysis of the tissue-specific composition of the human osteochondral junction in osteoarthritis: A pilot study.

This study investigates the influence of osteoarthritis (OA) disease severity on the bio-composition of the osteochondral junction at the human tibial plateau using Raman microspectroscopy. We specifically aim to analyze the spatial composition of mineralized osteochondral tissues, i.e., calcified cartilage (CC) and subchondral bone plate (SBP) from unfixed, hydrated specimens. We hypothesize that the mineralization of CC and SBP decreases in advanced OA. Twenty-eight cylindrical osteochondral samples (d = 4 mm) from tibial plateaus of seven cadaveric donors were harvested and sorted into three groups following histopathological grading: healthy (n = 5), early OA (n = 8), and advanced OA (n = 15). Raman spectra were subjected to multivariate cluster analyses to identify different tissues. Finally, the tissue-specific composition was analyzed, and the impact of OA was statistically evaluated with linear mixed models. Cluster analyses of Raman spectra successfully distinguished CC and SBP as well as a tidemark region and uncalcified cartilage. CC was found to be more mineralized and the mineral was more crystalline compared with SBP. Both tissues exhibited similar compositional changes as a function of histopathological OA severity. In early OA, the mineralization tends to increase, and the mineral contains fewer carbonate substitutions. Compared with early OA, mineral crystals are rich in carbonate while the overall mineralization decreases in advanced OA. This Raman spectroscopic study advances the methodology for investigating the complex osteochondral junction from native tissue. The developed methodology can be used to elucidate detailed tissue-specific changes in the chemical composition with advancing OA. STATEMENT OF SIGNIFICANCE: In this study, Raman microspectroscopy was utilized to investigate the influence of osteoarthritic degeneration on the tissue-specific biochemical composition of the human osteochondral junction. Multivariate cluster analyses allowed us to characterize subtle compositional changes in the calcified cartilage and subchondral bone plate as well as in the tidemark region. The compositional differences found between the calcified cartilage and subchondral bone plate in both organic and mineral phases will serve as critical benchmark parameters when designing biomaterials for osteochondral repair. We found tissue-specific changes in the mineralization and carbonate substitution as a function of histopathological OA severity. Our developed methodology can be used to investigate the metabolic changes in the osteochondral junction associated with osteoarthritis.