Proteoglycan Gel Research Articles

A hyperelastic biphasic fibre-reinforced model of articular cartilage considering distributed collagen fibre orientations: continuum basis, computational aspects and applications

Cartilage is a multi-phase material composed of fluid and electrolytes (68–85% by wet weight), proteoglycans (5–10% by wet weight), chondrocytes, collagen fibres and other glycoproteins. The solid phase constitutes an isotropic proteoglycan gel and a fibre network of predominantly type II collagen, which provides tensile strength and mechanical stiffness. The same two components control diffusion of the fluid phase, e.g. as visualised by diffusion tensor MRI: (i) the proteoglycan gel (giving a baseline isotropic diffusivity) and (ii) the highly anisotropic collagenous fibre network. We propose a new constitutive model and finite element implementation that focus on the essential load-bearing morphology: an incompressible, poroelastic solid matrix reinforced by an inhomogeneous, dispersed fibre fabric, which is saturated with an incompressible fluid residing in strain-dependent pores of the collagen–proteoglycan solid matrix. The inhomogeneous, dispersed fibre fabric of the solid further influences the fluid permeability, as well as an intrafibrillar portion that cannot be ‘squeezed out’ from the tissue. Using representative numerical examples on the mechanical response of cartilage, we reproduce several features that have been demonstrated experimentally in the cartilage mechanics literature.

Effectiveness of adsorbed film and gel layer in hydration lubrication as adaptive multimode lubrication mechanism for articular cartilage

With various daily activities, the effectiveness of adsorbed film formed on a gel-like layer at the uppermost superficial articular cartilage in natural synovial joints becomes important to control the friction and wear of articular cartilage in mixed or boundary lubrication regime as an adaptive multimode lubrication mechanism. Furthermore, in the case where the adsorbed film has been removed, the proteoglycan gel layer is expected to preserve low friction and protect against the wear of bulk cartilage tissue with an effective hydration lubrication mechanism. Besides, it is indicated that the biphasic lubrication plays an important role in lowering of friction in articular cartilage containing a large amount of water. At the present stage, however, the detailed relationship between adsorbed films and hydrated gel layers has not yet been elucidated. In this article, the frictional behaviours of articular cartilage on a glass plate were observed in the reciprocating tests with the restarting process after interruption and unloading. The lubricating effectiveness of adsorbed films in hyaluronate (HA) solutions was examined using intact and partially damaged cartilage specimens. The role of albumin and γ-globulin in relation to the surface conditions of gel layer is discussed. The restarting friction immediately after reloading became lower as a result of recovery of the effective interstitial fluid pressurization and hydration and adsorbed films have a significant effect on the frictional behaviour at local contacts. To clarify the molecular phenomena taking place under rubbing condition, in situ observations of the forming adsorbed film were conducted. The measurements were performed using the fluorescent staining method for protein and HA molecules at low contact pressures.

Articular Cartilage: Structure and Regeneration

Articular cartilage (AC) has no or very low ability of self-repair, and untreated lesions may lead to the development of osteoarthritis. One method that has been proven to result in long-term repair or isolated lesions is autologous chondrocyte transplantation. However, first generation of these cells' implantation has limitations, and introducing new effective cell sources can improve cartilage repair. AC provides a resilient and compliant articulating surface to the bones in diarthrodial joints. It protects the joint by distributing loads applied to it, so preventing potentially damaging stress concentrations on the bone. At the same time it provides a low-friction-bearing surface to enable free movement of the joint. AC may be considered as a visco- or poro-elastic fiber-composite material. Fibrils of predominantly type II collagen provide tensile reinforcing to a highly hydrated proteoglycan gel. The tissue typically comprises 70% water and it is the structuring and retention of this water by the proteoglycans and collagen that is largely responsible for the remarkable ability of the tissue to support compressive loads.

Imaging Articular Cartilage Tissue Using Atomic Force Microscopy (AFM): Figure 1.

Cartilage is a complex avascular tissue composed of cells ("chondrocytes") embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends. Atomic force microscopy (AFM) can be used to examine structure-function relationships of cartilage at both micrometer and nanometer scales. When imaged at the micrometer scale with microspheres, only the ECM and chondrocytes can be distinguished. Correspondingly, mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety. In this protocol, the corresponding AFM image and force map are used to reveal the distinct morphology of the collagen fibers and proteoglycan gel. Although, in principle, these experiments can be performed using any AFM, an AFM with tube scanners that have manual screws for tilting the sample is preferable because cartilage has macroscopically rough surface features. By manually tilting the probe over the sample, an optimal angle for tip approach can be achieved.

Micro- and Nanomechanical Analysis of Articular Cartilage by Indentation-Type Atomic Force Microscopy: Validation with a Gel-Microfiber Composite

As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilage's soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilage's lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage.

Roles of adsorbed film and gel layer in hydration lubrication for articular cartilage

To maintain low friction and low wear in natural synovial joints, adsorbed film formation on articular cartilage surface appears to play an important role in mixed or boundary lubrication regime where local direct contact occurs. Furthermore, the proteoglycan gel layer at the uppermost superficial zone in articular cartilage appears to play an important role in preserving low friction and low wear by the hydration lubrication mechanism even after removal of adsorbed film. However, the interaction or synergistic action between adsorbed film and hydrated film/surface has not yet been clarified. To examine the roles of adsorbed film and gel layer on articular cartilage surface in hydration lubrication, the changes in friction were observed in the reciprocating test of articular cartilage against a glass plate, at repeated rubbing including restarting after interrupting—unloading process. It is noticed that at restarting immediately after loading, hydration lubrication is expected to become effective. The lubricating roles of protein-adsorbed film in hydration lubrication are discussed on the basis of experimental results in both cases with and without surface gel layer. The comparison of albumin and γ -globulin is described in connection with the existence of a gel layer.

Microstructural Modeling of Collagen Network Mechanics and Interactions with the Proteoglycan Gel in Articular Cartilage

Cartilage matrix mechanical function is largely determined by interactions between the collagen fibrillar network and the proteoglycan gel. Although the molecular physics of these matrix constituents have been characterized and modern imaging methods are capable of localized measurement of molecular densities and orientation distributions, theoretical tools for using this information for prediction of cartilage mechanical behavior are lacking. We introduce a means to model collagen network contributions to cartilage mechanics based upon accessible microstructural information (fibril density and orientation distributions) and which self-consistently follows changes in microstructural geometry with matrix deformations. The interplay between the molecular physics of the collagen network and the proteoglycan gel is scaled up to determine matrix material properties, with features such as collagen fibril pre-stress in free-swelling cartilage emerging naturally and without introduction of ad hoc parameters. Methods are developed for theoretical treatment of the collagen network as a continuum-like distribution of fibrils, such that mechanical analysis of the network may be simplified by consideration of the spherical harmonic components of functions of the fibril orientation, strain, and stress distributions. Expressions for the collagen network contributions to matrix stress and stiffness tensors are derived, illustrating that only spherical harmonic components of orders 0 and 2 contribute to the stress, while orders 0, 2, and 4 contribute to the stiffness. Depth- and compression-dependent equilibrium mechanical properties of cartilage matrix are modeled, and advantages of the approach are illustrated by exploration of orientation and strain distributions of collagen fibrils in compressed cartilage. Results highlight collagen-proteoglycan interactions, especially for very small physiological strains where experimental data are relatively sparse. These methods for determining matrix mechanical properties from measurable quantities at the microscale (composition, structure, and molecular physics) may be useful for investigating cartilage structure-function relationships relevant to load-bearing, injury, and repair.

A model of unloaded human intervertebral disk based on NMR relaxation.

NMR relaxation rates were related to the composition of the nucleus pulposus from 11 and anulus fibrosus from six human intervertebral disks. Tissue water was proportional to glycosaminoglycan (GAG) and residue, the noncollagen, non-GAG portion of the dry weight (R2 = 0.74). The solid signal fraction depended on collagen and residue protons (R2 = 0.89). 1/T1 was proportional to collagen and residue (R2 = 0.97). T2 showed 2-4 components labeled A, B, C, and D, with means +/- standard deviations of 3.1 +/- 1.6, 17.5 +/- 9.5, 64 +/- 22, and 347 +/- 162 msec. Signal fractions of A and B depended on the collagen-associated water protons (R2 = 0.94 and 0.85), C on residue-associated water protons (R2 = 0.82), and D on GAG-associated water protons (R2 = 0.74). The data led to a model of disk architecture in which the collagen and residue were largely solid, forming distinct water compartments; the remaining water was present in a proteoglycan gel.

A model of unloaded human intervertebral disk based on NMR relaxation

NMR relaxation rates were related to the composition of the nucleus pulposus from 11 and anulus fibrosus from six human intervertebral disks. Tissue water was proportional to glycosaminoglycan (GAG) and residue, the noncollagen, non-GAG portion of the dry weight (R2= 0.74). The solid signal fraction depended on collagen and residue protons (R2= 0.89). 1/T1 was proportional to collagen and residue (R2= 0.97). T2 showed 2–4 components labeled A, B, C, and D, with means ± standard deviations of 3.1 ± 1.6, 17.5 ± 9.5, 64 ± 22, and 347 ± 162 msec. Signal fractions of A and B depended on the collagen-associated water protons (R2= 0.94 and 0.85), C on residue-associated water protons (R2= 0.82), and D on GAG-associated water protons (R2= 0.74). The data led to a model of disk architecture in which the collagen and residue were largely solid, forming distinct water compartments; the remaining water was present in a proteoglycan gel. Magn Reson Med 43:34–44, 2000. © 2000 Wiley-Liss, Inc.

The design of soft collagenous load-bearing tissues.

Tendon, articular cartilage and the human heel pad are all soft load-bearing collagenous tissues but are designed according to utterly different micromechanical principles. Tendon is (probably) a fibre-reinforced composite material. The mechanical properties of cartilage depend on osmotic pressure developed within an aqueous proteoglycan gel and resisted by tension in a collagenous network. The micromechanics of the heel pad have not previously been described quantitatively. Order-of-magnitude calculations are introduced to assess a model based on a fluid-filled cushion. The processes of biological design are illustrated by considering tendon. Structural design determines the tendon's cross-sectional area relative to that of its muscle and, hence, the maximum stress to which the tendon may be subjected in life. Stress-in-life varies widely between tendons. Material design includes the development of compressive stiffness in the regions where transverse loads arise. More generally, the fatigue quality of each tendon is adjusted to suit its stress-in-life. The correlation between fatigue quality and stress-in-life means that every tendon is subject, on average, to a comparable rate of fatigue damage. Homeostasis requires that routine repair can keep up with this rate of damage.

Influence of physical exercise on aging rats. III. Life-long exercise modifies the aging changes of the mechanical properties of limb muscle tendons

We have previously shown that long-term regular physical exercise has a systemic influence on the rat by slowing the aging of its connective tissues, measured as thermal stability and biomechanical properties of tail tendons. This paper analyses whether the properties of limb muscle tendons are influenced not only by the aging process and the systemic effects of exercise but also from direct mechanical stimuli from long-term physical exercise. Male Sprague-Dawley rats were trained in a treadmill from the age of 5 to 23 months. The effects of training on muscle tendons were analyzed with respect to biomechanical properties. Also, the viscoelastic activation energies for interactions between collagen and the proteoglycan gel as well as between collagen fibrils were measured. Finally the asymptotes from the creep curves were calculated in order to estimate the magnitude of the viscoelastic creep. The effects of aging were analyzed with respect to the same parameters by comparing the group of 23-month-old sedentary rats with a 5-month-old baseline group. The biomechanical parameters did not change significantly with physical exercise. Neither did the activation energies change, but the asymptotes of the creep curves decreased, showing that there was less viscoelastic creep. Aging rendered the tendons significantly stronger and stiffer, increased the energy-absorbing capacity and decreased the strain values. The activation energies did not change with aging, but the high creep curve asymptote for the flexor tendons decreased. We conclude that aging rendered both types of tendons stiffer, and decreased their strain values at breaking point. Aging also increased the stress value, the energy absorption and the dry weight for the flexor tendon. Further, while physical exercise has a systemic delaying effect on age changes in connective tissues, in tendons subjected to substantial mechanical loads this effect as measured with biomechanical methods is counteracted by the optimization process elicited by the same physical exercise.

Fibre reinforcing by collagen in cartilage and soft connective tissues.

Mechanical support in animals is performed by connective tissues. The soft tissues consist of collagen fibres embedded in a highly hydrated proteoglycan gel. By considering them as fibrous composite materials, a unifying theme can be found to explain their mechanical behaviour in terms of their structure and composition. Interactions between fibres and matrix are essential to their functioning in this way. Calculations are made of the maximum stress transfer per D-period required to enable collagen fibres of a given axial ratio to provide effective reinforcing. Weak non-specific interactions are shown to be sufficient. A mechanical function is proposed for type X collagen in the epiphyseal growth plate on the basis that it modifies and supplements the properties of the type II fibrils. This provides extra reinforcing and, hence, a greater stiffness to the cartilage to compensate for the reduced amount of extracellular matrix.

Effects of microgravity on the composition of the intervertebral disk.

The lumbar annuli of rats flown on COSMOS 2044 were compared with those of three control groups and a tail-suspension experimental model. The wet and dry weights of the annuli were significantly smaller (P less than 0.05) in the flight group than in three control groups. The collagen-to-proteoglycan ratio was significantly greater (P less than 0.001) in the flight group than in the three control groups, but there were no detectable changes in the relative proportions of type I and II collagen or in the number of pyridinoline cross-links. When the annuli were immersed in water for 2 h, more proteoglycans (P less than 0.001) leached from the annuli of flown rats than from the tissue of control animals, suggesting abnormal or smaller proteoglycans. Safranin-O indicated a normal spatial distribution of the proteoglycans within the annulus. Tail suspension did not affect the size of the annuli, but more proteoglycans (P less than 0.05) leached from the tissue of suspended animals than from the normal annuli. The reasons for smaller disks and the abnormal ratio between the fibrous collagenous network and the proteoglycan gel in the flight group are unknown at this time. It is, however, probable that these changes may affect the biomechanical functions of the annulus, although they may be temporary and totally reversible if injuries are avoided in the interim period.

Is classical consolidation theory applicable to articular cartilage deformation?

In this paper, classical consolidation theory has been used to investigate the time-dependent response of articular cartilage to static loading. An experimental technique was developed to measure simultaneously the matrix internal pressure and creep strain under conditions of one-dimensional consolidation. This is the first measurement of the internal stress state of loaded cartilage. It is demonstrated that under static compression the applied load is shared by the components of the matrix (i.e. water, the proteoglycans, and the collagen fibrillar meshwork), during which time a maximum hydrostatic excess pore pressure is developed as initial water exudation occurs. This pressure decays as water is further exuded from the matrix and effective consolidation begins with a progressive transfer of the applied stress from water to the collagen fibrils and proteoglycan gel. Consolidation is completed when the hydrostatic excess pore pressure is reduced to zero and the solid components sustain in full the applied load.

Tumour spheroid technology in cancer therapy research

GENERAL GROWTH PATTERN Spheroids have an outer layer of proliferating cells and an inside layer of mainly quiescent cells. In the central areas of spheroids with a diameter greater than 400-700 pm, there are often massive necrotic areas. This growth pattern is similar to that ofsolid tumour nodules containing proliferating cells close to the capillaries, quiescent cells next to these and necrotic areas at larger distances. Spheroid cells can be further divided into subpopulations as, for example, quiescent cells close to the necrotic centre which are irreversibly growth inhibited and quiescent cells closer to the surface that have the capacity to enter the cell cycle. The pattern is general although there are large cell-type dependent variations in growth rate, cell morphology and the thickness of the viable cell layers [l-7]. The spheroids contain an extensive extracellular matrix composed of, for example, fibronectin, laminin and collagen in a proteoglycan gel. There are differences between different types of spheroids also in this aspect and the differences correlate with differences in growth [8-l 11.

Biochemical and biophysical properties of the tectorial membrane (TM)

It was recently reported [I. Thalmann et al., ORL 48, 107–115 (1986)] that 40%–50% of the guinea pig TM consists of collagen. We now provide further rigorous proof that most of the collagen consists of type II, and that also the minor cartilage collagens, 1α, 2α, and 3α, as well as collagen type IX are present. Thus, the TM contains the same types of collagen as hyaline cartilage. Moreover, we have shown by quick-freeze, deep-etch 3-D electronmicroscopy (3-D-EM) that at least the upper part of the TM consists of a dense tangle of fibrils (∼20 nm) which are indistinguishable from the type II collagen fibrils of cartilage. These fibrils are embedded in a dense meshwork of anastomotic material that looks similar to the proteoglycan gel observed throughout cartilage but which differs from cartilage in that its constituent strands look relatively thick and variably puffy. Although the majority of the noncollagenous proteins are also glycoproteins, it cannot yet be decided whether and to what extent they contribute to a second type of major fibril or to the gel matrix. The presentation will include a description of the dry weight of the TM as a function of cochlear location and an estimate of the dry-to-wet weight ratio. [Work supported by NSF; We thank Dr. J. Heuser for the 3-D-EM preparations.]