Biorheology - Volume 41, issue 3-4

Authors: Guilak, Farshid | Awad, Hani A. | Fermor, Beverley | Leddy, Holly A. | Gimble, Jeffrey M.

Article Type: Research Article

Abstract: Tissue engineering is a promising therapeutic approach that uses combinations of implanted cells, biomaterial scaffolds, and biologically active molecules to repair or regenerate damaged or diseased tissues. Many diverse and increasingly complex approaches are being developed to repair articular cartilage, with the underlying premise that cells introduced exogenously play a necessary role in the success of engineered tissue replacements. A major consideration that remains in this field is the identification and characterization of appropriate sources of cells for tissue‐engineered repair of cartilage. In particular, there has been significant emphasis on the use of undifferentiated progenitor cells, or “stem” cells that …can be expanded in culture and differentiated into a variety of different cell types. Recent studies have identified the presence of an abundant source of stem cells in subcutaneous adipose tissue. These cells, termed adipose‐derived adult stem (ADAS) cells, show characteristics of multipotent adult stem cells, similar to those of bone marrow derived mesenchymal stem cells (MSCs), and under appropriate culture conditions, synthesize cartilage‐specific matrix proteins that are assembled in a cartilaginous extracellular matrix. The growth and chondrogenic differentiation of ADAS cells is strongly influenced by factors in the biochemical as well as biophysical environment of the cells. Furthermore, there is strong evidence that the interaction between the cells, the extracellular biomaterial substrate, and growth factors regulate ADAS cell differentiation and tissue growth. Overall, ADAS cells show significant promise for the development of functional tissue replacements for various tissues of the musculoskeletal system. Show more

Keywords: Articular cartilage, chondrocyte, pre‐adipocyte, stromal cell, osteoarthritis, collagen, proteoglycan

Citation: Biorheology, vol. 41, no. 3-4, pp. 389-399, 2004

Authors: Raimondi, Manuela T. | Boschetti, Federica | Falcone, Leonarda | Migliavacca, Francesco | Remuzzi, Andrea | Dubini, Gabriele

Article Type: Research Article

Abstract: This work examines the effect of perfusion on human mature articular chondrocytes cultured on synthetic biodegradable scaffolds (DegraPol). Human chondrocytes were isolated, seeded on the scaffolds and subjected to perfused culture at a flow rate of 0.5 ml/min, corresponding to an average inlet fluid velocity of 44 μm/s, with flow inversion every 1 minute. The flow was imposed at the construct surface in some constructs, it was forced through the construct thickness in other constructs and was absent in the static controls. We compared cell viability and morphology and we evaluated material properties of the constructs at 1 month of …culture. Thickness‐perfused constructs showed significantly higher material properties and roughly a two‐fold cell viability, when compared both to surface‐perfused constructs and to static controls. Chondrocytes maintained a phenotypic morphology in all experiments, probably favoured by a limited cell–scaffold interaction. Biosynthetic activity could be demonstrated only in the bioreactor‐cultured constructs. In this experimental model, a bi‐directional flow of culture medium was applied to the cells at a macroscopic level and computational modelling was used to quantify the fluid‐dynamic environment induced on the cells at a microscopic level. This method may be used to quantify the effects of fluid‐dynamic shear on the growth modulation of tissue‐engineered cartilage constructs, to potentially enhance tissue growth in vitro. Show more

Keywords: Tissue engineering, articular cartilage, mechanobiology, chondrocyte, bioreactor, computational fluid dynamics

Citation: Biorheology, vol. 41, no. 3-4, pp. 401-410, 2004

Authors: van Osch, Gerjo J.V.M. | Mandl, Erik W. | Jahr, Holger | Koevoet, Wendy | Nolst‐Trenité, Gilbert | Verhaar, Jan A.N.

Article Type: Research Article

Abstract: Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor‐site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer culture. Cell yield per gram of cartilage was twice as high for ear than for articular cartilage. Moreover, ear chondrocytes proliferated faster. Cell proliferation could be further stimulated by the use of serum‐free medium with Fibroblast Growth Factor 2 (FGF2) in stead of medium with 10% serum. To evaluate chondrogenic capacity, multiplied chondrocytes were suspended …in alginate and implanted subcutaneously in athymic mice. After 8 weeks the constructs demonstrated a proteoglycan‐rich matrix that contained collagen type II. Constructs of ear chondrocytes showed a faint staining for elastin. Quantitative RT‐PCR revealed that expression of collagen type II was 2‐fold upregulated whereas expression of collagen type I was 2‐fold down regulated in ear chondrocytes expanded in serum‐free medium with FGF2 compared to serum‐containing medium. Expression of alkaline phosphatase and collagen type X were low indicating the absence of terminal differentiation. We conclude that ear chondrocytes can be used as donor chondrocytes for cartilage tissue engineering. Furthermore, it may proof to be a promising alternative cell source to engineer cartilage for articular repair. Show more

Keywords: FGF2, dedifferentiation, elastin, articular

Citation: Biorheology, vol. 41, no. 3-4, pp. 411-421, 2004

Authors: Gigant‐Huselstein, C. | Hubert, P. | Dumas, D. | Dellacherie, E. | Netter, P. | Payan, E. | Stoltz, J.F.

Article Type: Research Article

Abstract: Chondrocytes use mechanical signals, via interactions with their environment, to synthesize an extracellular matrix capable to withstanding high loads. Most chondrocyte–matrix interactions are mediated via transmembrane receptors such as integrins or non‐integrins receptors (i.e. annexin V and CD44). The aim of this study was to analyze, by flow cytometry, the adhesion molecules (α5/β1 integrins and CD44) on rat chondrocytes seeded into 3D biosystem made of alginate and hyaluronate. These biosystems were submitted to mechanical stress by knocking the biosystems between them for 48 hours. The expression of type I and type II collagen was also evaluated. The results …of the current study showed that mechanical stress induced an increase of type II collagen production and weak variations of α5/β1 receptors expression no matter what biosystems. Moreover, our results indicated that hyaluronan receptor CD44 expression depends on extracellular matrix modifications. Thus, these receptors were activated by signals resulted from cell environment variations (HA addition and modifications owing to mechanical stress). It suggested that this kind of receptor play a crucial role in extracellular matrix homeostasis. Finally, on day 24, no dedifferentiation of chondrocytes was noted either in biosystems or under mechanical stress. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was type II, whatever biosystems. In conclusion, it appeared that the cells, under mechanical stress, maintained their phenotype. In addition, it seems that, on rat chondrocytes, α5/β1 integrins did not act as the main mechanoreceptor (as described for human chondrocytes). In return, hyaluronan receptor CD44 seems to be in relation with matrix composition. Show more

Citation: Biorheology, vol. 41, no. 3-4, pp. 423-431, 2004

Authors: Galois, L. | Freyria, A.M. | Grossin, L. | Hubert, P. | Mainard, D. | Herbage, D. | Stoltz, J.F. | Netter, P. | Dellacherie, E. | Payan, E.

Article Type: Research Article

Abstract: Lesions of articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Replacement of articular defects in joints has assumed greater importance in recent years. This interest results in large part because cartilage defects cannot adequately heal themselves. Many techniques have been suggested over the last 30 years, but none allows the regeneration of the damaged cartilage, i.e. its replacement by a strictly identical tissue. In the first generation of techniques, relief of pain was the main concern, which could be provided by techniques in which cartilage was replaced by …fibrocartilage. Disappointing results led investigators to focus on more appropriate bioregenerative approaches using transplantation of autologous cells into the lesion. Unfortunately, none of these approaches has provided a perfect final solution to the problem. The latest generation of techniques, currently in the developmental or preclinical stages, involve biomaterials for the repair of chondral or osteochondral lesions. Many of these scaffolds are designed to be seeded with chondrocytes or progenitor cells. Among natural and synthetic polymers, collagen‐ and polysaccharide‐based biomaterials have been extensively used. For both these supports, studies have shown that chondrocytes maintain their phenotype when cultured in three dimensions. In both types of culture, a glycosaminoglycan‐rich deposit is formed on the surface and in the inner region of the cultured cartilage, and type II collagen synthesis is also observed. Dynamic conditions can also improve the composition of such three‐dimensional constructs. Many improvements are still required, however, in a number of key aspects that so far have received only scant attention. These aspects include: adhesion/integration of the graft with the adjacent native cartilage, cell‐seeding with genetically‐modified cell populations, biomaterials that can be implanted without open joint surgery and combined therapies, aimed at disease modification, pain relief and reduction of inflammation. Show more

Keywords: Cartilage repair, surgical techniques, tissue engineering, collagen, polysaccharide‐based biomaterials, chondrocyte

Citation: Biorheology, vol. 41, no. 3-4, pp. 433-443, 2004

Authors: Seidel, J.O. | Pei, M. | Gray, M.L. | Langer, R. | Freed, L.E. | Vunjak‐Novakovic, G.

Article Type: Research Article

Abstract: One approach to functional tissue engineering of cartilage is to utilize bioreactors to provide environmental conditions that stimulate chondrogenesis in cells cultured on biomaterial scaffolds. We report the combined use of a three‐dimensional in vitro model and a novel bioreactor with perfusion of culture medium and mechanical stimulation in long‐term studies of cartilage development and function. To engineer cartilage, scaffolds made of a non‐woven mesh of polyglycolic acid (PGA) were seeded with bovine calf articular chondrocytes, cultured for an initial 30‐day period under free swelling conditions, and cultured for an additional 37 day period in one of the three groups: …(1) free‐swelling, (2) static compression (on 24 h/day, strain control, static offset 10%), and (3) dynamic compression (on 1 h/day; off 23 h/day; strain control, static offset 2%, dynamic strain amplitude 5%; frequency 0.3 Hz). Constructs were sampled at timed intervals and assessed with respect to structure, biochemical composition, and mechanical function. Mechanical simulation had little effect on the compositions, morphologies and on mechanical properties of construct interiors discs, but it resulted in distincly different properties of the peripheral rings and face sides. Contructs cultured with mechanical loading maintained their cylindrical shape with flat and parallel top and bottom surfaces, and retained larger amounts of GAG. The modular bioreactor system with medium perfusion and mechanical loading can be utilized to define the conditions of cultivation for functional tissue engineering of cartilage. Show more

Keywords: Chondrogenesis, bioreactor, loading, fluid flow, glycosaminoglycans, collagen

Citation: Biorheology, vol. 41, no. 3-4, pp. 445-458, 2004

Authors: Dumas, D. | Gaborit, N. | Grossin, L. | Riquelme, B. | Gigant‐Huselstein, C. | de Isla, N. | Gillet, P. | Netter, P. | Stoltz, J.F.

Article Type: Research Article

Abstract: Spectral and multiphoton imaging is the preferred approach for non‐invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross‐talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and …allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM‐FRET). Show more

Keywords: GFP variants, glycophorin, FRET, lifetime imaging, spectral, chondrocytes, erythrocytes

Citation: Biorheology, vol. 41, no. 3-4, pp. 459-467, 2004

Authors: Martin, James A. | Brown, Thomas | Heiner, Anneliese | Buckwalter, Joseph A.

Article Type: Research Article

Abstract: Joint injuries frequently lead to progressive joint degeneration that causes the clinical syndrome of post‐traumatic osteoarthritis. The pathogenesis of osteoarthritis remains poorly understood, but patient age is a significant risk factor for progressive joint degeneration. We have found that articular cartilage chondrocytes show strong evidence of senescence with increasing age, including synthesis of smaller more irregular aggrecans; increased expression of lysosomal beta‐galactosidase and telomere erosion; and decreased proteoglycan synthesis, response to the anabolic cytokine IGF‐I, proliferative capacity, and mitochondrial function. These observations help explain the strong association between age and joint degeneration, but they do not explain how joint injury …increases the risk of joint degeneration in younger individuals. We hypothesized that excessive loading of articular surfaces due to acute joint trauma or post‐traumatic joint instability, incongruity or mal‐alignment increases release of reactive oxygen species, and that the increased oxidative stress on chondrocytes accelerates chondrocyte senescence thereby decreasing the ability of the cells to maintain or restore the tissue. To test this hypothesis, we exposed human articular cartilage chondrocytes from young adults to mechanical and oxidative stress. We found that shear stress applied to cartilage explants in a triaxial pressure vessel increased release of reactive oxygen species and oxidative stress induced chondrocyte senescence (as measured by expression of lysosomal beta‐galactosidase, nuclear and mitochondrial DNA damage and decreased mitochondrial function). These observations support the hypothesis that joint injury accelerates chondrocyte senescence and that this acceleration plays a role in the joint degeneration responsible for post‐traumatic osteoarthritis. Show more

Citation: Biorheology, vol. 41, no. 3-4, pp. 479-491, 2004

Authors: Brandt, Kenneth D. | Smith, Gerald N. | Myers, Stephen L.

Article Type: Research Article

Abstract: We previously reported that intraarticular injections of hyaluronan (HA), administered prophylactically to dogs in whom knee osteoarthritis had been induced by transection of the anterior cruicate ligament, did not significantly modify the intraarticular pathology but decreased the proteogylcan concentration of the articular cartilage by as much as 30%. Because the cartilage proteoglycan concentration is directly related to the stiffness of the tissue, these results raised the possibility that intraarticular HA therapy could exacerbate OA. In the present study, using a different HA formulation, with a longer interval between intraarticular HA injection and examination of joint tissues, we found that neither …prophylactic nor therapeutic administration of HA had an effect on the severity of OA pathology, the magnitude of vertical ground reaction forces generated by the unstable hind limb (a surrogate for joint pain), or the cartilage proteoglycan concentration. The data suggest that the suppression of proteoglycan synthesis induced by HA is temporary and fully reversible and that HA injections do not result in overloading of the OA extremity. A significant correlation was noted between the severity of chondropathy and the magnitude of the vertical ground reaction forces generated by the unstable limb. Show more

Keywords: Intra‐articular injection, gait, limb loading

Citation: Biorheology, vol. 41, no. 3-4, pp. 493-502, 2004

Authors: Grossin, Laurent | Etienne, Stéphanie | Gaborit, Nadège | Pinzano, Astrid | Cournil‐Henrionnet, Christel | Gerard, Catherine | Payan, Elisabeth | Netter, Patrick | Terlain, Bernard | Gillet, Pierre

Article Type: Research Article

Abstract: The aim of this work was to determine whether Hsp70 overexpression via proteasome inhibitor MG132 was able to protect chondrocytes towards mono‐iodoacetate (MIA) cytotoxicity both in vitro and in vivo. In vitro, overexpression of Hsp70 via MG132 was significantly able to protect chondrocytes from MIA toxicity (MTT/LDH analyses). Hsp70 essentially mediated this chondroprotective effect as demonstrated by antisense strategy. In vivo, chondrocytic overexpression of Hsp70, after a preventive intra‐articular injection of MG132 in rat knee, was sufficient to decrease the severity of OA‐induced MIA lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of Hsp70, through proteasome inhibition, could …be an interesting tool in protecting chondrocytes from cellular injuries, either necrotic or apoptotic in nature, and thus might be a novel chondroprotective modality in rat experimental OA. Show more

Citation: Biorheology, vol. 41, no. 3-4, pp. 521-534, 2004

Authors: Gouttenoire, Jérôme | Valcourt, Ulrich | Ronzière, Marie‐Claire | Aubert‐Foucher, Elisabeth | Mallein‐Gerin, Frédéric | Herbage, Daniel

Article Type: Research Article

Abstract: In osteoarthritic cartilage, chondrocytes are able to present heterogeneous cellular reactions with expression and synthesis of the (pro)collagen types characteristic of prechondrocytes (type IIA), hypertrophic chondrocytes (type X), as well as differentiated (types IIB, IX, XI, VI) and dedifferentiated (types I, III) chondrocytes. The expression of type IIA procollagen in human osteoarthritic cartilage support the assumption that OA chondrocytes reverse their phenotype towards a chondroprogenitor phenotype. Recently, we have shown that dedifferentiation of mouse chondrocytes induced by subculture was associated with the alternative splicing of type II procollagen pre‐mRNA with a switch from the IIB to the IIA form. In …this context, we demonstrated that BMP‐2 favours expression of type IIB whereas TGF‐β1 potentiates expression of type IIA induced by subculture. These data reveal the specific capability of BMP‐2 to reverse the program of chondrocyte dedifferentiation. This interesting feature needs to be tested with human chondrocytes since cell amplification is required for the currently used autologous chondrocyte transplantation. Show more

Keywords: Chondrocyte phenotype, type IIA and IIB procollagen, BMP‐2, TGF‐β1, cartilage repair

Citation: Biorheology, vol. 41, no. 3-4, pp. 535-542, 2004

Authors: Stoltz, J.‐F.

Article Type: Other

Citation: Biorheology, vol. 41, no. 3-4, pp. 565-565, 2004

Authors: Millward‐Sadler, S.J. | Wright, M.O. | Flatman, P.W. | Salter, D.M.

Article Type: Research Article

Abstract: Extracellular nucleotides have been shown to have diverse effects on chondrocyte function, generally acting via P2 purinoceptors. We have previously shown that mechanical stimulation at 0.33 Hz of normal human chondrocyte cultures causes cellular hyperpolarisation, while chondrocytes derived from osteoarthritic (OA) cartilage depolarise. Experiments have been undertaken to establish whether ATP is involved in the response of the chondrocyte to mechanical stimulation. Chondrocytes, isolated from normal and OA cartilage obtained, with consent, from human knee joints following surgery, were cultured in non‐confluent monolayer. Cells were mechanically stimulated at 0.33 Hz for 20 minutes at 37°C in the presence or …absence of inhibitors of ATP signalling, or were stimulated by the addition of exogenous ATP or derivatives, and electrophysiological measurements recorded. Samples of medium bathing the cells were collected before and after mechanical stimulation, and the concentration of ATP in the cell medium was measured. Total RNA was extracted from cultured chondrocytes, reverse‐transcribed and used for RT‐PCR with primers specific for P2Y2 purinoceptors. ATP, UTP 2‐methylthioadenosine and αβ‐methylene adenosine 5′‐triphosphate all induced a hyperpolarisation response in normal human articular chondrocytes. No significant change was observed in the membrane potentials of chondrocytes isolated from OA cartilage following the addition of these nucleotides to the medium. In normal chondrocytes, the hyperpolarisation induced by ATP was blocked by the presence of apamin, indicating the involvement of small‐conductance calcium‐activated potassium channels. Following mechanical stimulation of normal chondrocytes, an increase was observed in ATP concentration in the cell culture medium bathing the cells. The presence within the culture medium of suramin or pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) prior to and during the period of mechanical stimulation abolished the hyperpolarisation response in normal chondrocytes. The presence of mRNA for P2Y2 purinoceptors was demonstrated in both normal and OA chondrocytes by RT‐PCR. These results suggest that ATP has a role in the response of normal chondrocytes to mechanical stimulation, via P2Y2 purinoceptors. This response appears to be different in chondrocytes derived from OA cartilage, and may be important in the progression of this disease. Show more

Keywords: Osteoarthritis, mechanical stimulation, signal transduction, purinoceptor

Citation: Biorheology, vol. 41, no. 3-4, pp. 567-575, 2004

Authors: Lima, Eric G. | Mauck, Robert L. | Han, Shelley H. | Park, Seonghun | Ng, Kenneth W. | Ateshian, Gerard A. | Hung, Clark T.

Article Type: Research Article

Abstract: Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue‐engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long‐term culture environment, one can enhance the development of mechanical properties in chondrocyte‐seeded agarose constructs. As these gel constructs begin to achieve material properties similar to …that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage‐like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral (“gel alone”) constructs see a largely homogenous field of mechanical signals, osteochondral (“gel bone”) constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute. Show more

Keywords: Functional tissue engineering, deformational loading, articular cartilage, osteochondral constructs, finite element models

Citation: Biorheology, vol. 41, no. 3-4, pp. 577-590, 2004

Authors: Lammi, Mikko J.

Article Type: Research Article

Abstract: It is well known that physiological forces are essential for the maintenance of normal composition and structure of articular cartilage. Although some of the mechanisms of mechanotransduction are known today, there are certainly many others left unrevealed. In order to understand the complicated systems present in articular cartilage, we have to bring together the data from all fields of cartilage mechanobiology. The 3rd Symposium on Mechanobiology of Cartilage and Chondrocyte was a good effort towards that goal.

Citation: Biorheology, vol. 41, no. 3-4, pp. 593-596, 2004

Authors: Stoltz, J.‐F.

Article Type: Other

Citation: Biorheology, vol. 41, no. 3-4, pp. 597-597, 2004

Authors: Luo, Zong‐Ping | Sun, Yu‐Long | Fujii, Tadashi | An, Kai‐Nan

Article Type: Research Article

Abstract: Type II collagen and hyaluronan are the two major components of extracellular molecules in cartilage and play an important role in mechanical functions of extracellular matrix. Currently, their mechanical properties have been investigated only at the gross‐level. In this study, the mechanical properties of single type II collagen and hyaluronan molecules were directly measured using optical tweezers technique. The persistence length was found to be 11.2±8.4 nm in type II collagen and 4.5±1.2 nm in hyaluronan. This result suggested that type II collagen is stiffer than hyaluronan at the individual molecule level, which supports the general concept that collagen is …responsible for resisting tensile force. The experimental system developed here also provides a powerful tool for quantifying mechanical properties of extracellular matrix at the single molecule level. Show more

Keywords: Type II collagen, hyaluronan, optical tweezers, persistence length, mechanical properties

Citation: Biorheology, vol. 41, no. 3‐4, pp. 247-254, 2004

Authors: Salter, D.M. | Wright, M.O. | Millward‐Sadler, S.J.

Article Type: Research Article

Abstract: Mechanical forces influence articular cartilage structure by regulating chondrocyte activity. Mechanical stimulation results in activation of an α5β1 integrin dependent intracellular signal cascade involving focal adhesion kinase and protein kinase C, triggering the release of interleukin‐4 from the cell. In normal HAC the response to physiological mechanical stimulation is characterised by increased levels of aggrecan mRNA and a decrease in levels of mRNA for matrix metalloproteinase 3 (MMP‐3), the net result of which would be to maintain and optimise cartilage structure and function. This protective/anabolic response is not seen when chondrocytes from osteoarthritic cartilage are subjected to an identical mechanical …stimulation regime. Following the observation that the neurotransmitter substance P is involved in chondrocyte mechanotransduction the present study was undertaken to establish potential roles for glutamate receptors in the control of chondrocyte mechanical responses. Using immunohistochemistry and RTPCR normal and OA chondrocytes are shown to express NR1 and NR2a subunits of the NMDA receptor. Addition of NMDA receptor agonists to chondrocytes in primary culture resulted in changes in membrane potential consistent with expression of functional receptors. NMDA receptor antagonists inhibited the hyperpolarisation response of normal chondrocytes to mechanical stimulation but had no effect on the depolarisation response of osteoarthritic chondrocytes to mechanical stimulation. These studies indicate that at least one subset of the NMDA receptor family of molecules is expressed in cartilage and may have important modulatory effects on mechanotransduction and cellular responses following mechanical stimulation. Indeed the results suggest that there is an alteration of NMDA receptor signalling in OA chondrocytes, which may be critical in the abnormal response of OA chondrocytes to mechanical stimulation. Thus NMDA receptors appear to be involved in the regulation of human articular chondrocyte responses to mechanical stimulation, and in OA, mechanotransduction pathways may be modified as a result of altered activation and function of these receptors. Show more

Citation: Biorheology, vol. 41, no. 3‐4, pp. 273-281, 2004

Authors: Bailey, Allen J. | Mansell, Jason P. | Sims, Trevor J. | Banse, Xavier

Article Type: Research Article

Abstract: The subchondral bone has long been known to thicken in osteoarthritis. However, recent evidence has demonstrated that the turnover of the bone is increased several fold, and further suggests that the thickening occurs prior to degradation of the articular cartilage, indicating that it plays a role in the pathogenesis of osteoarthritis. The mechanical and biochemical properties of the subchondral bone are therefore of particular interest in any attempt to determine the nature of the factors initiating osteoarthritis. We have shown that the subchondral bone collagen of the femoral head possessed a 20‐fold increase in turnover, as assessed by procollagen …rate of synthesis and metalloproteinase degradation, and a 25% decrease in mineralisation. This increased metabolism and high lysyl hydroxylation leads to narrower and weaker fibres. Additionally the phenotypic expression of the osteoblasts is modified to produce increasing proportions of type I homotrimer in addition to the normal type I heterotrimer, which further reduces the mechanical strength of the bone. Overall, the narrow immature collagen fibres, the reduction in pyrrole cross‐linking, decreased mineralisation, and increased amounts of type I homotrimer, all contribute to a weakening of the mechanical properties of the subchondral bone. Show more

Keywords: Subchondral bone, osteoarthritis, collagen, cross‐linking, metabolism, biomechanics

Citation: Biorheology, vol. 41, no. 3‐4, pp. 349-358, 2004

Authors: Day, J.S. | van der Linden, J.C. | Bank, R.A. | Ding, M. | Hvid, I. | Sumner, D.R. | Weinans, H.

Article Type: Research Article

Abstract: Osteoarthritis is a chronic joint disease with pathological changes in the articulating cartilage and all other tissues that occupy the joint. Radin and coworkers have suggested the involvement of subchondral bone in the disease process. However, evidence for an essential role in the etiology has never been proven. Recent studies showing reduced chemical and mechanical properties of subchondral bone in various stages of the disease have invigorated interest in the role of subchondral bone in the development and progression of the disease. The current study showed that the concept of bone adaptation might explain subchondral stiffening, a process where subchondral …bone becomes typically sclerotic in osteoarthritis. In addition, we report reduced mechanical matrix tissue properties as well as an increase in denatured collagen content. In conclusion, although osteoarthritic bone tissue contains increased denatured collagen and has reduced matrix mechanical properties, the widely accepted concept of subchondral stiffening is compatible with the process of normal bone adaptation. Show more

Citation: Biorheology, vol. 41, no. 3‐4, pp. 359-368, 2004

Authors: Angoulvant, D. | Clerc, A. | Benchalal, S. | Galambrun, C. | Farre, A. | Bertrand, Y. | Eljaafari, A.

Article Type: Research Article

Abstract: Mesenchymal stem cells (MSC) fail to induce allogeneic responses in mixed lymphocyte reaction assays. Because MSC express HLA class I molecules, here we investaged whether they could be recognized as allogeneic targets by cytolytic T lymphocytes (CTL). With this aim, CTL precursor (CTLp) frequencies were measured following stimulation of T cells with either allogeneic mononuclear cells (MNC) or MSC originated from the same human bone marrow donor. Lysis of MSC was measured at day 10 of culture in standard chromium release assays. In addition, allogeneic PHA blast T cells or B‐EBV lymphoblastoid cell lines (LCLs) generated from the same donor …were used as positive controls of lysis. Our results showed that when allogeneic MNC were used to stimulate T cells, a high CTLp frequency was detected towards MSC targets. However, when MSC were used as stimulators, CTLp frequencies were markedly altered whatever the targets used, i.e.: MSC, PHA blast T cells or EBV‐B LCLs. Moreover, when graded concentrations of MSC were added together with MNC upon stimulation of alloreactive T cells, we observed a dose‐dependent decrease in CTLp frequencies towards MSC targets. This inhibition of MSC lysis was partially overcomed by adding exogenous rh‐IL‐2 from the beginning of cultures. In addition, this suppressive effect was totally reproduced when, instead of MSC, supernatant harvested from MSC cultures was added to allogeneic MNC, upon stimulation of alloreactive T cells. In conclusion, our results demonstrate that MSC which can be recognized as targets by pre‐activated alloreactive CTLs, may be able to suppress differentiation of CTL precursors into CTL effectors through secretion of suppressive factors. Show more

Citation: Biorheology, vol. 41, no. 3‐4, pp. 469-476, 2004

Authors: Schaefer, Dirk B. | Wendt, David | Moretti, Matteo | Jakob, Marcel | Jay, Gregory D. | Heberer, Michael | Martin, Ivan

Article Type: Research Article

Abstract: Cartilage integration in vivo does not occur, such that even cartilage fissures do not heal. This could be due not only to the limited access of chondrocytes to the wound, but also to exogenous factors. In this paper, we tested the hypothesis that lubricin, a lubricating protein physiologically present in the synovial fluid, reduces the integrative cartilage repair capacity. Disk/ring composites of bovine articular cartilage were prepared using concentric circular blades and cultured for 6 weeks with or without treatment with 250 μg/ml lubricin applied three times per week. Following culture, the percentage of contact area between the disks and …the rings, as assessed by light microscopy, were equal in both groups. The adhesive strength of the integration interface, as assessed by push‐out mechanical tests, was markedly and significantly lower in lubricin‐treated specimens (2.5 kPa) than in the controls (28.7 kPa). Histological observation of Safranin‐O stained cross‐sections confirmed the reduced integration in the lubricin treated composites. Our findings suggest that the synovial milieu, by providing lubrication of cartilage surfaces, impairs cartilage–cartilage integration. Show more

Citation: Biorheology, vol. 41, no. 3‐4, pp. 503-508, 2004

Authors: Morel, V. | Quinn, T.M.

Article Type: Research Article

Abstract: The short‐term responses of articular cartilage to mechanical injury have important implications for prevention and treatment of degenerative disease. Cell and matrix responses were monitored for 11 days following injurious compression of cartilage in osteochondral explants. Injury was applied as a single ramp compression to 14 MPa peak stress at one of three strain rates: 7×10−1 , 7×10−3 or 7×10−5 s−1 . Responses were quantified in terms of the appearance of macroscopic matrix cracks, changes in cell viability, and changes in cartilage wet weights. Loading at the highest strain rate resulted in acute cell death near the superficial …zone in association with cracks, followed over the 11 days after compression by a gradual increase in cell death and loss of demarcation between matrix zones containing viable versus nonviable cells. In contrast, loading at the lowest strain rate resulted in more severe, nearly full‐depth cell death acutely, but with no apparent worsening over the 11 days following compression. Between days 4 and 11, all mechanically injured explants significantly increased in wet weight, suggesting loss of matrix mechanical integrity independent of compression strain rate. Results demonstrate that short‐term responses of cartilage depend upon the biomechanical characteristics of injurious loading, and suggest multiple independent pathways of mechanically‐induced cell death and matrix degradation. Modifications to an existing fiber‐reinforced poroelastic finite element model were introduced and the model was used for data interpretation and identification of microphysical events involved in cell and matrix injury. The model performed reasonably well at the slower strain rates and exhibited some capacity for anticipating the formation of superficial cracks during injurious loading. However, several improvements appear to be necessary before such a model could reliably be used to draw upon in vitro experimental results for prediction of injurious loading situations in vivo. Show more

Citation: Biorheology, vol. 41, no. 3‐4, pp. 509-519, 2004

Authors: Henrotin, Y. | Deberg, M. | Dubuc, J.‐E. | Quettier, E. | Christgau, S. | Reginster, J.‐Y.

Article Type: Research Article

Abstract: This paper describes two new immunoassays for a peptide of the triple helix of type II collagen (Coll 2‐1) and its nitrated form (Coll 2‐1 NO2 ). In healthy subjects aged between 20 and 65 years old, Coll 2‐1 and Coll 2‐1 NO2 levels in serum were in means 125.13±3.71 and 0.16±0.08 nmol/l, respectively. These levels did not significantly vary with age. However, up to 45 years of age, Coll 2‐1 NO2 levels in women were significantly higher than in men. In patients with knee osteoarthritis (OA), Coll 2‐1 in serum was found to be elevated compared to …healthy controls (267.45±26.42 nmol/l vs 126.78±6.61 nmol/l). Further, we have demonstrated that an increase of the urinary levels of Coll 2‐1 or Coll 2‐1 NO2 over 1 year was predictive of joint space narrowing progression in OA patients. In conclusion, these preliminary results indicate that Coll 2‐1 could be a predictive marker of knee OA progression. Show more

Keywords: Ageing, osteoarthritis, biological markers, nitric oxide, oxygen

Citation: Biorheology, vol. 41, no. 3‐4, pp. 543-547, 2004

Authors: Martin, G. | Bogdanowicz, P. | Domagala, F. | Ficheux, H. | Pujol, J.‐P.

Article Type: Research Article

Abstract: In the present report, we show that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL‐1β (10 ng/ml) by an increased DNA binding activity of NF‐κB and AP‐1 transcription factors. Incubation of the cells with 10−5 M rhein for 24 h was found to reduce this activity, particularly in the case of AP‐1. Mitogen activated kinases (ERK‐1 and ERK‐2) were activated by exposure of the chondrocytes to 1‐h treatment with IL‐1β. This effect was greater in hypoxia (3% O2 ) than in normoxia (21% O2 ). Rhein was …capable of reducing the IL‐1β‐stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady‐state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24‐h treatment with 10−5 M rhein. This stimulating effect was also observed in the presence of IL‐1β, suggesting that the drug could prevent or reduce the IL‐1β‐induced inhibition of extracellular matrix synthesis. IL‐1‐induced collagenase (MMP1) expression was significantly decreased by rhein in the same conditions. In conclusion, rhein can effectively inhibit the IL‐1‐activated MAPK pathway and the binding of NF‐κB and AP‐1 transcription factors, two key factors involved in the expression of several pro‐inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti‐osteoarthritic properties of rhein and its disease‐modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level. Show more

Keywords: Cartilage, chondrocytes, interleukin‐1, osteoarthritis, rhein

Citation: Biorheology, vol. 41, no. 3‐4, pp. 549-561, 2004