A fully coupled poroelastic reactive-transport model of cartilage

Cartilage maintains its integrity in a hostile mechanical environment. This task is made more difficult because cartilage has no blood supply, and so nutrients and growth factors need to be transported greater distances than normal to reach cells several millimetres from the cartilage surface. The chondrocytes embedded within the extracellular matrix (ECM) are essential for maintaining the mechanical integrity of the ECM, through a balance of degradation and synthesis of collagen and proteoglycans. A chondrocyte senses various chemical and mechanical signals in its local microenvironment, responding by appropriate adaption of the local ECM. Clearly a 'systems understanding' of cartilage behaviour is of critical importance in developing an integrated understanding of both normal and abnormal physiology of cartilage. In a series of papers, we have developed a reactive-transport porous-media model to investigate the coupled processes of growth factor transport, mechanical deformation and fluid flow, and in this paper, we extend the model to include biosynthesis and degradation of matrix molecules. The model is validated using three independent experimental data sets, it being found that a single set of parameters described the experimental results remarkably well. The model is then employed to make predictions about changes in proteoglycan content under a variety of conditions. This model may prove useful in predicting the behaviour of tissue engineering constructs, or predicting the outcome of repair processes in cartilage.     

The effect of continuous culture on the growth and structure of tissue‐engineered cartilage

The use of bioreactors for cartilage tissue engineering has become increasingly important as traditional batch‐fed culture is not optimal for in vitro tissue growth. Most tissue engineering bioreactors rely on convection as the primary means to provide mass transfer; however, convective transport can also impart potentially unwanted and/or uncontrollable mechanical stimuli to the cells resident in the construct. The reliance on diffusive transport may not necessarily be ineffectual as previous studies have observed improved cartilaginous tissue growth when the constructs were cultured in elevated volumes of media. In this study, to approximate an infinite reservoir of media, we investigated the effect of continuous culture on cartilaginous tissue growth in vitro. Isolated bovine articular chondrocytes were seeded in high density, 3D culture on Millicell? filters. After two weeks of preculture, the constructs were cultivated with or without continuous media flow (5–10 μL/min) for a period of one week. Tissue engineered cartilage constructs grown under continuous media flow significantly accumulated more collagen and proteoglycans (increased by 50–70%). These changes were similar in magnitude to the reported effect of through‐thickness perfusion without the need for large volumetric flow rates (5–10μL/min as opposed to 240–800 μL/min). Additionally, tissues grown in the reactor displayed some evidence of the stratified morphology of native cartilage as well as containing stores of intracellular glycogen. Future studies will investigate the effect of long‐term continuous culture in terms of extracellular matrix accumulation and subsequent changes in mechanical function. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Proteoglycan deposition around chondrocytes in agarose culture: construction of a physical and biological interface for mechanotransduction in cartilage

With a view towards the development of methods for cartilage tissue engineering, matrix deposition around individual chondrocytes was studied during de novo matrix synthesis in agarose suspension culture. At a range of times in culture from 2 days to 1 month (long enough for cartilage-like material properties to begin to emerge), pericellular distributions of proteoglycan and matrix protein deposition were measured by quantitative autoradiography, while matrix accumulation and cell volumes were estimated by stereological methods. Consistent with previous work, tissue-average rates of matrix synthesis generally decreased asymptotically with time in culture, as de novo matrix accumulated. Cell-scale analysis revealed that this evolution was accompanied by a transition from predominantly pericellular matrix (within a few microm from the cell membrane) deposition early in culture towards proteoglycan and protein deposition patterns more similar to those observed in cartilage explants at later times. This finding may suggest a differential recruitment of different proteoglycan metabolic pools as matrix assembly progresses. Cell volumes increased with time in culture, suggestive of alterations in volume regulatory processes associated with changes in the microphysical environment. Results emphasize a pattern of de novo matrix construction which proceeds outward from the pericellular matrix in a progressive fashion. These findings provide cell-scale insight into the mechanisms of assembly of matrix proteins and proteoglycans in de novo matrix, and may aid in the development of tissue engineering methods for cartilage repair.     

Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of approximately 6.25 microm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm(-1)/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.     

Perlecan displays variable spatial and temporal immunolocalisation patterns in the articular and growth plate cartilages of the ovine stifle joint

Perlecan is a modular heparan sulphate and/or chondroitin sulphate substituted proteoglycan of basement membrane, vascular tissues and cartilage. Perlecan acts as a low affinity co-receptor for fibroblast growth factors 1, 2, 7, 9, binds connective tissue growth factor and co-ordinates chondrogenesis, endochondral ossification and vascular remodelling during skeletal development; however, relatively little is known of its distribution in these tissues during ageing and development. The aim of the present study was to immunolocalise perlecan in the articular and epiphyseal growth plate cartilages of stifle joints in 2-day to 8-year-old pedigree merino sheep. Perlecan was prominent pericellularly in the stifle joint cartilages at all age points and also present in the inter-territorial matrix of the newborn to 19-month-old cartilage specimens. Aggrecan was part pericellular, but predominantly an extracellular proteoglycan. Perlecan was a prominent component of the long bone growth plates and displayed a pericellular as well as a strong ECM distribution pattern; this may indicate a so far unrecognised role for perlecan in the mineralisation of hypertrophic cartilage. A significant age dependant decline in cell number and perlecan levels was evident in the hyaline and growth plate cartilages. The prominent pericellular distribution of perlecan observed indicates potential roles in cell-matrix communication in cartilage, consistent with growth factor signalling, cellular proliferation and tissue development.     

Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of ∼6.25 μm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm⁻¹/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.     

Perlecan, the "jack of all trades" proteoglycan of cartilaginous weight-bearing connective tissues

Perlecan is a ubiquitous proteoglycan of basement membrane and vascularized tissues but is also present in articular cartilage, meniscus and intervertebral disc, which are devoid of basement membrane and predominantly avascular. It is a prominent pericellular proteoglycan in the transitory matrix of the cartilaginous rudiments that develop into components of diarthrodial joints and the axial skeleton, and it forms intricate perichondrial vessel networks that define the presumptive articulating surfaces of developing joints and line the cartilage canals in cartilaginous rudiments. Such vessels have roles in the nutrition of the expanding cell numbers in the developing joint. Perlecan sequesters a number of growth factors pericellularly (FGFs, PDGF, VEGF and CTGF) and through these promotes cell signalling, cell proliferation and differentiation. Perlecan also interacts with a diverse range of extracellular matrix proteins, stabilising and organising the ECM, and promoting collagen fibrillogenesis. Perlecan is a prominent pericellular component of mesenchymal cells from their earliest developmental stages through to maturation, forming cell-cell and cell-ECM interconnections that are suggestive of a role in mechanosensory processes important to tissue homeostasis.     

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Persistence of cartilage proteoglycan and link protein during matrix-induced endochondral bone development: An immunofluorescent study

Monospecific antibodies to cartilage proteoglycan monomer and link protein were employed with immunofluorescence microscopy to determine the tissue distribution of these constituents during matrix-induced endochondral bone development. Subcutaneous implantation of demineralized diaphyseal bone matrix resulted in new endochondral bone formation. On Day 3, the implant consisted of mesenchymal tissue which did not contain any demonstrable cartilage-related proteoglycan or link protein. With the onset of early chondrogenesis on Day 5, cartilage proteoglycan monomer and link protein were first localized together in the cartilage matrix, particularly around chondrocytes in territorial sites. Progressively more staining around cells was observed at Days 7 and 9. On Day 9, when mineralization was first observed, there was no evidence of a net loss of these molecules prior to mineralization of the cartilage matrix. On Day 11 and thereafter, bone formation was observed by appositional growth on calcified cartilage spicules. Whereas the osteoblasts and bone matrix were devoid of any staining for cartilage proteoglycan and link components, the residual, partly mineralized cartilage spicules still reacted with antibodies to cartilage proteoglycan monomer and link protein in territorial sites, but in reduced amounts, indicating a loss of these molecules associated with a loss of hypertrophic chondrocytes. Since mineral prevented the access of Fab' antibody subunits, demineralization after fixation was routinely employed. The results reveal that cartilage proteoglycan monomer and link protein are present around chondrocytes in hyaline cartilage during the early stages of endochondral bone formation and that there is no net loss of these molecules prior to mineralization of this cartilage matrix as was previously thought.     

Immunohistochemical localization of fibronectin and chondronectin in canine articular cartilage

We compared the distribution of fibronectin and chondronectin within the matrix of canine articular cartilage. Fibronectin was found throughout the matrix as well as pericellularly. In contrast, chondronectin was observed predominantly associated with the cell or pericellular matrix. Interactions of these molecules with matrix components in the pericellular matrix probably differs, however, since concentrations of hyaluronidase which prevented detection of pericellular fibronectin allowed detection of chondronectin. Chondronectin and fibronectin were detected in osteoarthritic cartilage as well as in disease-free cartilage. Penetration of biotinylated fibronectin into cartilage from the external medium occurred only in osteoarthritic cartilage and proceeded only from the articular surface. Disease-free cartilage appeared to maintain a barrier to fibronectin penetration from the articular surface which was sustained even after the proteoglycan content was markedly depleted by incubation of cartilage with catabolin or lipopolysaccharide. In cartilage that was proteoglycan-depleted, the only detectable penetration of external fibronectin was from the cut surface.     

Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min(-1)) and gradually increasing (0.075-0.2 mL min(-1)) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0-4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min(-1). GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.     

Squeeze-film lubrication of the human ankle joint with synovial fluid filtrated by articular cartilage with the superficial zone worn out

A squeeze-film lubrication model of the human ankle joint in standing that takes into account the fluid transport across the articular surface is presented. Articular cartilage is a biphasic mixture of the ideal interstitial fluid and an elastic permeable isotropic homogeneous intrinsically incompressible matrix. The simple homogeneous model for articular cartilage models the case of early osteoarthritis, when the intact superficial zone of the normal articular cartilage, much stiffer in tension than the bulk material, has been already disrupted or worn out. The calculations indicate for this case that in normal approach motion the lubricating fluid film is quickly depleted and turned into a synovial gel film that is supposed to serve as a boundary lubricant if sliding motion follows     

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.     

Protective effects of polydeoxyribonucleotides on cartilage degradation in experimental cultures

The capacity of cartilage self‐regeneration is considered to be limited. Joint injuries often evolve in the development of chronic wounds on the cartilage surface. Such lesions are associated with articular cartilage degeneration and osteoarthritis. Re‐establishing a correct micro/macro‐environment into damaged joints could stop or prevent the degenerative processes. This study investigated the effect of polydeoxyribonucleotides (PDRNs) on cartilage degradation in vitro and on cartilage extracted cells. The activities of matrix metalloproteinases 2 and 9 were measured in PDRN‐treated cells and in controls at days 0 and 30 of culture. Human nasal cartilage explants were cultured, and the degree of proteoglycan degradation was assessed by measuring the amount of glycosaminoglycans released into the culture medium. The PDRN properties compared with controls were tested on cartilage tissues to evaluate deposition of extracellular matrix. Chondrocytes treated with PDRNs showed a physiological deposition of extracellular matrix (aggrecan and type II collagen: Western blot, IFA, fluorescence activated cell sorting, Alcian blue and safranin O staining). PDRNs were able to inhibit proteoglycan degradation in cartilage explants. The activities of matrix metalloproteinases 2 and 9 were reduced in all PDRN‐treated samples. Our results indicate that PDRNs are suitable for a long‐term cultivation of in vitro cartilage and have therapeutic effects on chondrocytes by protecting cartilage. Copyright © 2012 John Wiley & Sons, Ltd.     

Dynamic compression augments interstitial transport of a glucose-like solute in articular cartilage

Solute transport through the extracellular matrix is essential for cellular activities in articular cartilage. Increased solute transport via fluid convection may be a mechanism by which dynamic compression stimulates chondrocyte metabolism. However, loading conditions that optimally augment transport likely vary for different solutes. To investigate effects of dynamic loading on transport of a bioactive solute, triangular mechanical loading waveforms were applied to cartilage explants disks while interstitial transport of a fluorescent glucose analog was monitored. Peak-to-peak compression amplitudes varied from 5-50% and frequencies varied from 0.0006-0.1 Hz to alter the spatial distribution and magnitude of oscillatory fluid flow. Solute transport was quantified by monitoring accumulation of fluorescence in a saline bath circulated around the explant. Individual explants were subjected to a series of compression protocols, so that effects of loading on solute desorption could be observed directly. Maximum increases in solute transport were obtained with 10-20% compression amplitudes at 0.1 Hz; similar loading protocols were previously found to stimulate chondrocyte metabolism in vitro. Results therefore support hypotheses relating to increased solute transport as a mediator of the cartilage biological response to dynamic compression, and may have application in mechanical conditioning of cartilage constructs for tissue engineering.     

Role of pericellular matrix in development of a mechanically functional neocartilage

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.     

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.     

Cartilage electromechanics--I. Electrokinetic transduction and the effects of electrolyte pH and ionic strength

Articular cartilage contains a high fixed charge density under physiological conditions associated primarily with the ionized proteoglycan molecules of the extracellular matrix. Oscillatory compression of cartilage using physiological loads produces electrical potentials that have been shown previously to be the result of an electrokinetic (streaming) transduction mechanism. We have now observed two additional electromechanical phenomena not previously seen in cartilage or other soft tissues: 'streaming current' and 'current-generated stress'. Sinusoidal mechanical compression induced a sinusoidal streaming current density through cartilage disks when the Ag/AgCl electrodes that were used to compress the cartilage were shorted together externally. Conversely, a sinusoidal current density applied to the tissue generated a sinusoidal mechanical stress within the tissue. Both these phenomena were found to be consistent with the same electrokinetic transduction mechanism responsible for the streaming potential. Changes in the measured streaming potential response that resulted from modification of bath ionic strength and pH have provided additional insights into the molecular origins of these transduction processes. Finally, we have now observed streaming potentials in living cartilage maintained in organ culture, as well as in previously frozen tissue.