Cartilage: Multiscale Structure and Biomechanical Properties

Macromolecules, 2021

Glycosaminoglycans (GAGs) are molecules that govern the load-bearing and frictional properties of cartilage and the lubricating properties of synovial fluid of joints. Most GAGs in the body form proteoglycans (PGs), and the dominant PG in cartilage is the bottlebrush-shaped aggrecan, comprised of a protein core decorated by GAG side chains consisting mainly of chondroitin sulfate (CS) and keratan sulfate. Additionally, hyaluronic acid (HA) induces aggrecan to self-assemble into complexes having up to 100 aggrecan molecules attached to each HA chain "backbone". Here, we report the rheological properties of CS, HA, aggrecan, and aggrecan−HA complexes in water and salt solutions. We find that CS solutions are viscous liquids, while HA solutions exhibit viscoelasticity and shear thinning. Specifically, in the case of HA, the storage modulus G′ exceeds the loss modulus G″ at high frequencies (ω) while the reverse is true at low ω. Thus, the rheologies of CS and HA exhibit somewhat distinct viscoelastic properties. Aggrecan, on the other hand, shows a rheology consistent with an incipient "weak gel" in which G′ and G″ cross at a specific ω and follow a power-law scaling over a wide ω range. Moreover, aggrecan samples do not attain an observable plateau in the viscosity under steady shear and low shear rates. Finally, the complexation of aggrecan and HA over a period of 48 h results in a slow evolution ("aging") in measured rheological properties, with G′ at low ω progressively increases in time, while G″ remains essentially unchanged. This suggests that aggrecan and HA gradually form a macroscopic network in which molecular complexes act as physical cross-links.

Journal of Structural Biology, 2003

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.

ACS nano, 2017

Articular cartilage is a natural biomaterial whose structure at the micro- and nanoscale is critical for healthy joint function and where degeneration is associated with widespread disorders such as osteoarthritis. At the nanoscale, cartilage mechanical functionality is dependent on the collagen fibrils and hydrated proteoglycans that form the extracellular matrix. The dynamic response of these ultrastructural building blocks at the nanoscale, however, remains unclear. Here we measure time-resolved changes in collagen fibril strain, using small-angle X-ray diffraction during compression of bovine and human cartilage explants. We demonstrate the existence of a collagen fibril tensile pre-strain, estimated from the D-period at approximately 1-2%, due to osmotic swelling pressure from the proteoglycan. We reveal a rapid reduction and recovery of this pre-strain which occurs during stress relaxation, approximately 60 s after the onset of peak load. Furthermore, we show that this reducti...

Biophysical Journal, 2010

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca 2þ -dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of l p ¼ 0.31 5 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of t ¼ 7.9 5 4.9 s and a reaction bond length of x b ¼ 0.31 5 0.08 nm. Whereas the x b -value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.

Journal of Biomechanics, 2010

This study presents direct experimental evidence for assessing the electrostatic and nonelectrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage. Immature and mature bovine cartilage samples were tested in unconfined compression and their depth-dependent equilibrium compressive modulus was determined using strain measurements with digital image correlation analysis. The electrostatic contribution was assessed by testing samples in isotonic and hypertonic saline; the combined contribution was assessed by testing untreated and proteoglycan-depleted samples. Though it is well recognized that proteoglycans contribute significantly to the compressive stiffness of cartilage, results demonstrate that the combined electrostatic and non-electrostatic contributions may add up to more than 98% of the modulus, a magnitude not previously appreciated. Of this contribution, about two-thirds arises from electrostatic effects. The compressive modulus of the proteoglycan-depleted cartilage matrix may be as low as 3 kPa, representing less than 2% of the normal tissue modulus; experimental evidence also confirms that the collagen matrix in digested cartilage may buckle under compressive strains, resulting in crimping patterns. Thus, it is reasonable to model the collagen as a fibrillar matrix that can only sustain tension. This study also demonstrates that residual stresses in cartilage do not arise exclusively from proteoglycans, since cartilage remains curled relative to its in situ geometry even after proteoglycan depletion. These increased insights into the structure-function relationships of cartilage can lead to improved constitutive models and a better understanding of the response of cartilage to physiological loading conditions.

Biophysical Journal, 2008

Here it is reported that aggrecan, the highly negatively charged macromolecule in the cartilage extracellular matrix, undergoes Ca 21 -mediated self-adhesion after static compression even in the presence of strong electrostatic repulsion in physiological-like solution conditions. Aggrecan was chemically end-attached onto gold-coated planar silicon substrates and goldcoated microspherical atomic force microscope probe tips (end radius R % 2.5 mm) at a density (;40 mg/mL) that simulates physiological conditions in the tissue (;20-80 mg/mL). Colloidal force spectroscopy was employed to measure the adhesion between opposing aggrecan monolayers in NaCl (0.001-1.0 M) and NaCl 1 CaCl 2 ([Cl À ] ¼ 0.15 M, [Ca 21 ] ¼ 0 -75 mM) aqueous electrolyte solutions. Aggrecan self-adhesion was found to increase with increasing surface equilibration time upon compression (0-30 s). Hydrogen bonding and physical entanglements between the chondroitin sulfate-glycosaminoglycan side chains are proposed as important factors contributing to aggrecan self-adhesion. Self-adhesion was found to significantly increase with decreasing bath ionic strength (and hence, electrostatic double-layer repulsion), as well as increasing Ca 21 concentration due to the additional ion-bridging effects. It is hypothesized that aggrecan self-adhesion, and the macromolecular energy dissipation that results from this self-adhesion, could be important factors contributing to the self-assembled architecture and integrity of the cartilage extracellular matrix in vivo.

The Biochemical journal, 1974

Ordered conformations of proteoglycan-hyaluronic acid aggregates in the intercellular matrix in cartilage were observed by X-ray diffraction. The sodium salt form of three samples, (a) aggregated proteoglycan, (b) disaggregated proteoglycan and (c) reconstituted disaggregated proteoglycan, give essentially similar X-ray fibre-type diffraction photographs. The patterns correlate with the chondroitin 4-sulphate component and can be interpreted as twofold helical conformations, similar to that observed previously for the free acid form of chondroitin 4-sulphate (Isaac & Atkins, 1973). The information takes us one step nearer the situation found in cartilage in vivo.

Scientific Reports, 2020

Articular cartilage is a load-bearing tissue found in animal and human joints. it is a composite gel-like material in which a fibrous collagen network encapsulates large proteoglycan assemblies that imbibe fluid and "inflate" the network. Here we describe a composite hydrogel consisting of a cross-linked polyvinyl alcohol matrix filled with poly(acrylic acid) microparticles that mimics functional properties and biomechanical behavior of cartilage. the swelling and mechanical behaviors of this biomimetic model system are strikingly similar to that of human cartilage. the development of synthetic composite gel-based articular cartilage analog suggests new avenues to explore material properties, and their change in disease and degeneration, as well as novel strategies for developing composite tissueengineered cartilage constructs for regenerative medicine applications. This manuscript describes a predictive composite hydrogel model of cartilage. A novel aspect of the model is the incorporation of the pre-stress, which has been largely overlooked in previous tissue models. Pre-stress is present even in the absence of external loading, and it defines the load-bearing ability of cartilage. We demonstrate that the swelling and mechanical behavior of our biomimetic model system reproduces remarkably well the behavior of healthy and osteoarthritic human cartilage. From a biological perspective, articular cartilage is a thin connective tissue (2 to 4 mm thick) that caps the ends of bones at joints. Cartilage extracellular matrix (ECM) consists mainly of proteoglycans (PGs), collagens, water, and ions. The most abundant macromolecule in cartilage ECM is Type II collagen, which is organized into fibrils that form a meshwork at larger length scales. The major PG in cartilage is the bottlebrush-shaped aggrecan that "inflates" the collagen matrix . Intracellularly synthesized aggrecan molecules are secreted into the ECM, where they aggregate to form a secondary bottlebrush with hyaluronic acid (HA) stabilized by a link protein . The osmotic swelling pressure of the aggrecan-HA assemblies inflates the surrounding collagen matrix (Fig. ). At swelling equilibrium, the tissue is prestressed, conferring unique mechanical properties on cartilage. At equilibrium, although the total swelling pressure is zero, the collagen matrix is in tension. In diseases, such as osteoarthritis, the lubricating and load-bearing ability is reduced, however, the mechanisms that lead to loss of cartilage's key biomechanical properties are still poorly understood. From a polymer science perspective, cartilage is a composite load-bearing gel, consisting of a relatively stiff, fibrous collagen network that encapsulates large, negatively charged (anionic) proteoglycan (PG) polymer assemblies that exhibit a hierarchical bottlebrush organization. These PG polyelectrolytes draw water in osmotically, causing this phase to swell against the collagen network phase that confines them, increasing the overall stiffness of the cartilage tissue. Under a compressive loading, fluid is expelled from the tissue; after unloading it recovers its original shape and volume. The resistance of cartilage to such deformation and volume changes defines its load-bearing capacity. In degenerative joint diseases (e.g., osteoarthritis) cartilage structure is progressively damaged. Changes in the stiffness of the collagen matrix and/or loss of PGs strongly affect the biomechanical properties (e.g., load-bearing capacity) of the tissue 9,10 . Although a qualitative picture of cartilage behavior being explained by the interplay between the swelling of the PGs and the restraint imposed by the collagen network was first proposed by Ogston 1,2 , a deeper polymer chemistry and physics-based understanding of the determinants of cartilage's key functional properties, such as its load-bearing ability, has eluded adequate description.

MRS Proceedings, 2014

ABSTRACTCartilage is a complex biological tissue that exhibits gel-like behavior. Its primary biological function is providing compressive resistance to external loading and nearly frictionless lubrication of joints. In this study, we model cartilage extracellular matrix using a biomimetic system. We demonstrate that poly(vinyl) alcohol (PVA) hydrogels are robust biomaterials exhibiting mechanical and swelling properties similar to that of cartilage extracellular matrix. A comparison is made between the macroscopic behavior of PVA gels and literature data reported for cartilage.

The Journal of Physical Chemistry B, 2021

Structural stability of various collagen containing biomaterials such as bones and cartilage is still a mystery. Despite spectroscopic development of several decades, the detailed mechanism of collagen interaction with citrate in bones and glycosaminoglycans (GAGS) in cartilage extracellular matrix (ECM) in its native state is unobservable. We present a significant advancement to probe the collagen interactions with citrate and GAGSin ECM of native bone and cartilage along with specific/non-specific interactions inside collagen assembly at the nanoscopic level through natural abundance dynamic nuclear polarization (DNP) based solid-state nuclear magnetic resonance (ssNMR) spectroscopy. The detected molecular level interactions between citrate-collagen and glycosaminoglycan (GAGs)-collagen inside the native bone and cartilage matrix and other backbone and side-chain interactions in the collagen assembly are responsible for the structural stability and other biomechanical properties of these important class of biomaterial.