Biorheology - Volume 45, issue 3-4

Authors: Stoltz, J.F.

Article Type: Other

DOI: 10.3233/BIR-2008-0501

Citation: Biorheology, vol. 45, no. 3-4, pp. 189-189, 2008

Authors: Gratz, Kenneth R. | Wong, Benjamin L. | Bae, Won C. | Sah, Robert L.

Article Type: Research Article

Abstract: Focal damage to articular cartilage is commonly found in symptomatic knees and may contribute to patient discomfort and progressive cartilage degeneration. The objective of this study was to quantify changes in cartilage intra-tissue strain and sliding occurring near a focal defect. Pairs of human osteochondral blocks were compressed by 20% of the total cartilage thicknesses, and tissue deformation was recorded by video microscopy. Then, a single, full-thickness defect was created in one block from each pair, blocks were allowed to re-swell, and the pairs were retested. Stained nuclei, acting as fiducial markers, were tracked by digital image correlation and used …to calculate cartilage strains and surface displacement. With intact samples, axial strain decreased with depth, as is typical of cartilage, and relatively little sliding occurred between surfaces. With defect samples, axial compression of cartilage at the defect rim rose by ~30%, shear in the opposing tissue increased 10-fold to ~0.15, and local sliding was elevated to >50 μm. In vivo, tissue near a defect likely experiences increased overall compression, magnifying these observed in vitro effects. Excessive strains may contribute to cell death, matrix damage, or accelerated wear, and repair efficacy may depend on the ability to alleviate adverse mechanical conditions. Show more

Keywords: Osteoarthritis, biomechanics, cartilage damage, cartilage deformation

DOI: 10.3233/BIR-2008-0475

Citation: Biorheology, vol. 45, no. 3-4, pp. 193-207, 2008

Authors: Scott, John E.

Article Type: Research Article

Abstract: Animal shapes are maintained by connective tissue extracellular matrices (ECMs). ECM shapes depend on keeping collagen fibrils in the right places, held by regular frequent proteoglycan (PG) bridges attached at specific sites. The PGs carry anionic glycosaminoglycan (AGAG) ‘strings’ that span and determine interfibrillar distances, thus holding us together. I called these repeating structures ‘shape modules’. The strings are aggregated antiparallel chains of dermochondan, keratan and chondroitan sulphates (DS, KS and CS); stabilised by hydrophobic and H-bonds. Shape modules are elastic. AGAG/AGAG interactions break under stress and reform when the stress is removed and/or they contain an elastic sugar, L-iduronate …(in DS). Cartilage ECMs are also based on shape modules. Depots therein of aggrecan, the large PG which carries many chains of CS and KS, imbibe water, thereby exerting swelling pressure. External pressure forces this water into the elastic shape modules, from whence it is returned post compression. Cartilage anisotropic responses (along and at right angles to shape module axes) to compressive and tensile stresses are now interpretable. Degradation of shape modules in osteoarthrosis reduces these responses. Inability to hold collagen fibrils together results in imbibition of excess water, fissuring and erosion, characteristic of this condition. Show more

Keywords: Shape modules, collagen fibrils, proteoglycans, decoran, aggrecan, osteoarthrosis

DOI: 10.3233/BIR-2008-0477

Citation: Biorheology, vol. 45, no. 3-4, pp. 209-217, 2008

Authors: Haudenschild, D.R. | D'Lima, D.D. | Lotz, M.K.

Article Type: Research Article

Abstract: The signal transduction mechanisms in chondrocytes that recognize applied forces and elicit the appropriate biochemical cellular responses are not well characterized. A current theory is that the actin cytoskeleton provides an intracellular framework onto which mechanosensation mechanisms are assembled. The actin cytoskeleton is linked to the extracellular matrix at multi-protein complexes called focal adhesions, and evidence exists that focal adhesions mediate the conversion of external physical forces into appropriate biochemical signal transduction events. The Rho GTPases affect the arrangement of actin cytoskeletal structures, and enhance the formation of focal adhesions, which link the cytoskeleton to the extracellular matrix. A …major effector pathway downstream of Rho is the activation of Rho kinase (ROCK), which phosphorylates and activates Lim kinase, which in turn phosphorylates and inhibits the actin-depolymerizing protein cofilin. The objectives of this study were threefold: first, to quantify the actin reorganization in response to dynamic compression of agarose-embedded chondrocytes. Second, to test whether Rho kinase is required for the actin cytoskeletal reorganization induced by dynamic compression. Third, to test whether dynamic compression alters the intracellular localization of Rho kinase and actin remodeling proteins in chondrocytes. Dynamic compression of agarose-embedded chondrocytes induced actin cytoskeletal remodeling causing a significant increase in punctate F-actin structures. Rho kinase activity was required for these cytoskeletal changes. Dynamic compression increased the amount of phosphorylated Rho kinase. The chemokine CCL20 and inducible nitric oxide synthase (iNOS) were the most highly upregulated genes by dynamic compression and this response was reduced by the Rho kinase inhibitors. In conclusion, we show that dynamic compression induces changes in the actin cytoskeleton of agarose-embedded chondrocytes, and we establish methodology to quantify these changes. Furthermore, we show that Rho kinase activity is required for this actin reorganization and gene expression induced by dynamic compression. Show more

Keywords: ROCK, gene expression, LIM kinase, CCL20, NOS2A

DOI: 10.3233/BIR-2008-0499

Citation: Biorheology, vol. 45, no. 3-4, pp. 219-228, 2008

Authors: Akanji, O.O. | Lee, D.A. | Bader, D.A.

Article Type: Research Article

Abstract: Endogenous electrical activity has been detected in articular cartilage. It has previously been suggested that the associated electrical currents and potentials are important to the mechanotransduction processes in cartilage. The present study investigates the effects of direct current on cell proliferation and matrix synthesis, using the well established 3D chondrocyte – agarose model system. Bovine chondrocytes isolated from metacarpalphalangeal joints were seeded in agarose constructs and exposed to a current density of 4 mA/cm2 for 6 h, a magnitude and period which was shown to maintain cell viability. The influence of the optimized electric stimulus was assessed by …protein incorporation and mRNA measurements, using radiolabels and real-time QPCR, respectively. Results indicated no systematic influences of electrical current on protein synthesis, cell proliferation and mRNA expression levels. These data suggest that both the mode of stimulation and the model system are critical for the in vitro modulation of chondrocyte metabolism. Show more

Keywords: Electric stimulation, chondrocyte, proteoglycan, collagen

DOI: 10.3233/BIR-2008-0473

Citation: Biorheology, vol. 45, no. 3-4, pp. 229-243, 2008

Authors: Anghelina, Mirela | Sjostrom, Danen | Perera, Priyangi | Nam, Jin | Knobloch, Thomas | Agarwal, Sudha

Article Type: Research Article

Abstract: Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or injured joints, but also for promoting a homeostatic equilibrium in healthy joints. Human joints provide the pivot points and physiological hinges essential for ambulation and movement to the body, and it is this mobility that in return promotes the health of the joints. But how mobilization regulates the joint microenvironment at the molecular level has remained enigmatic for many years. Recent advances in joint biomechanics and molecular approaches have facilitated an enriched understanding of how joints operate. Consequently, the mechanisms active during joint inflammation that lead …to arthritic conditions, both in vivo in animal models, and in vitro at cell and tissue levels, have become increasingly detailed and defined. These efforts have produced mounting evidences supporting the premise that biomechanical signals play a fundamental role in both the etiopathogenesis of arthritic diseases and in the physiological restoration of joints. This report aims to summarize current peer-reviewed literature and available experimental data to explain how the signals generated by mechanical forces/joint mobilization generate beneficial effects on inflamed articular cartilage, and to propose the basis for using appropriate physical therapies for the optimal benefit to the patient suffering from joint associated injuries. Show more

Keywords: Cartilage, chondrocytes, mechanical strain, NF-κB, signal transduction, inflammation

DOI: 10.3233/BIR-2008-0472

Citation: Biorheology, vol. 45, no. 3-4, pp. 245-256, 2008

Authors: Chowdhury, T.T. | Akanji, O.O. | Salter, D.M. | Bader, D.L. | Lee, D.A.

Article Type: Research Article

Abstract: Interleukin-1β (IL-1β) induces the release of nitric oxide (· NO) and prostaglandin E2 (PGE2 ) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1β upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating · NO and PGE2 release in IL-1β stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng·ml−1 IL-1β …with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of α5β1 integrin signalling and p38 MAPK activation in mediating the release of · NO and PGE2 in response to both IL-1β and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of · NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1β enhanced · NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1β enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of · NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2. Show more

Keywords: P38 MAPK, nitric oxide, PGE_2, mechanotransduction, chondrocyte

DOI: 10.3233/BIR-2008-0474

Citation: Biorheology, vol. 45, no. 3-4, pp. 257-274, 2008

Authors: Holledge, M. Maruo | Millward-Sadler, S.J. | Nuki, G. | Salter, D.M.

Article Type: Research Article

Abstract: The importance of biomechanical forces in regulating normal chondrocyte metabolism is well established and the mechanisms whereby mechanical forces are transduced into biochemical responses by chondrocytes are beginning to be understood. Previous studies have indicated that cyclical mechanical stimulation induces increased aggrecan gene expression in normal but not osteoarthritic chondrocytes in monolayer. It remains unclear, however, whether these effects on gene expression are associated with changes in proteoglycan production and whether any changes in proteoglycan expression is dependent on integrins or integrin associated proteins. Normal and osteoarthritic articular chondrocytes in monolayer were exposed to 0.33 Hz mechanical stimulation for …20 min in the absence or presence of function modifying anti-integrin antibodies. Following stimulation GAG and proteoglycan (PG) synthesis was assessed by DMMB assay and western blotting. Mechanical stimulation of normal chondrocytes resulted in increased GAG synthesis that was blocked by the presence of antibodies to α5 and αVβ5 integrins and CD47. Electrophoretic patterns of PGs released from normal chondrocytes following mechanical stimulation showed an increase in newly-synthesized aggrecan that was not fragmented or degraded. Chondrocytes from osteoarthritic cartilage showed lower levels of GAG production when compared to normal chondrocytes and synthesis was not influenced by mechanical stimulation. These studies show that chondrocytes derived from normal and OA cartilage have different matrix production responses to mechanical stimulation and suggest previously unrecognised roles for αVβ5 integrin in regulation of chondrocyte responses to biomechanical stimulation. Show more

Keywords: GAG, cartilage, CD47, monolayer

DOI: 10.3233/BIR-2008-0476

Citation: Biorheology, vol. 45, no. 3-4, pp. 275-288, 2008

Authors: Nishida, Takashi | Maeda, Azusa | Kubota, Satoshi | Takigawa, Masaharu

Article Type: Research Article

Abstract: Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be …mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration. Show more

Keywords: Mechanobiology, meniscus cells, chondrocytes, hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), aggrecan, mechanical stress

DOI: 10.3233/BIR-2008-0478

Citation: Biorheology, vol. 45, no. 3-4, pp. 289-299, 2008

Authors: Gosset, Marjolaine | Berenbaum, Francis | Levy, Arlette | Pigenet, Audrey | Thirion, Sylvie | Cavadias, Simeon | Jacques, Claire

Article Type: Research Article

Abstract: NOTE: Several parts of this article were copied verbatim or close to verbatim from an article that was published in Arthritis Research & Therapy, 2006, 8:R135, doi:10.1186/ar2024. No acknowledgement of, or reference to, the previously published article in Arthritis Research & Therapy was made at the time of publication of the article in Biorheology. In this updated version of the article all the copied parts are highlighted. An Erratum has been published in Biorheology 49(4) (2012), 299. Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2 ) …is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2 . Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis. Show more

Keywords: PGE_2, mPGES-1, COX-2, 15-PGDH, MMP, murine cartilage, mechanical stress, osteoarthritis

DOI: 10.3233/BIR-2008-0494

Citation: Biorheology, vol. 45, no. 3-4, pp. 301-320, 2008

Authors: Piltti, Juha | Häyrinen, Jukka | Karjalainen, Hannu M. | Lammi, Mikko J.

Article Type: Research Article

Abstract: For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of …1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells. Show more

Keywords: Mechanobiology, cellular stretching, signal transduction, phosphorylation

DOI: 10.3233/BIR-2008-0481

Citation: Biorheology, vol. 45, no. 3-4, pp. 323-335, 2008

Authors: Boschetti, Federica | Peretti, Giuseppe M.

Article Type: Research Article

Abstract: Osteoarthritis (OA) is a disease affecting articular cartilage and the underlying bone, resulting from many biological and mechanical interacting factors which change the extracellular matrix (ECM) and cells and lead to increasing levels of cartilage degeneration, like softening, fibrillation, ulceration and cartilage loss. The early diagnosis of the disease is fundamental to prevent pain, further tissue degeneration and reduce hospital costs. Although morphological modifications can be detected by modern non-invasive diagnostic techniques, they may not be evident in the early stages of OA. The mechanical properties of articular cartilage are related to its composition and structure and are sensitive to …even small changes in the ECM that could occur in early OA. The aim of the present study was to compare the mechanical properties of healthy and OA cartilage using a combined experimental–numerical approach. Experimental assessments consisted of step wise confined and unconfined compression and tension stress relaxation tests on disks (for compression) or strips (for tension) of cartilage obtained from human femoral heads discarded from the operating room after total hip replacement. The numerical model was based on the biphasic theory and included the tension–compression non-linearity. Considering OA samples vs normal samples, the static compressive modulus was 55–68% lower, the permeability was 60–80% higher, the dynamic compressive modulus was 59–64% lower, the static tension modulus was 72–83% lower. The model successfully simulated the experimental tests performed on healthy and OA cartilage and was used in combination with the experimental tests to evaluate the role of different ECM components in the mechanical response of normal and OA cartilage. Show more

Keywords: Articular cartilage, poroelastic modeling, biomechanical tests, osteoarthritis

DOI: 10.3233/BIR-2008-0479

Citation: Biorheology, vol. 45, no. 3-4, pp. 337-344, 2008

Authors: Leskinen, Jarkko J. | Karjalainen, Hannu M. | Olkku, Anu | Hynynen, Kullervo | Mahonen, Anitta | Lammi, Mikko J.

Article Type: Research Article

Abstract: It is well documented that low intensity pulsed ultrasound can be clinically used to accelerate bone fracture healing. Additionally, in vitro studies have shown that ultrasound can, for instance, increase mineralization, collagen production and alkaline phosphatase activity in osteoblasts. Despite the extensive research on the subject, the exact mechanism of ultrasound effect on bone cell gene regulation has not yet been deduced. In this study, we made an effort to reveal the features of genome-wide transcriptional response of osteoblast-type cells to ultrasound. MG-63 osteoblastic cell transcriptome was analyzed with whole genome microarray either 6 or 24 h after 30 min …long exposure to 1.035 MHz pulsed ultrasound with three different acoustic pressures. Special attention was paid to the experimental design to minimize thermal effects and unwanted reflections of ultrasound. Microarray analysis suggested that ultrasound affects the genes involved with cellular membranes, and regulation of transcription as well. Several plasma membrane solute carriers were also regulated by ultrasound. It also changed the transcript level of several transcription factors belonging to the zinc finger proteins. However, ultrasound did not clearly promote genes involved with osteoblast differentiation. Show more

Keywords: Bone cell, gene expression, transcriptome, ultrasound stimulation

DOI: 10.3233/BIR-2008-0480

Citation: Biorheology, vol. 45, no. 3-4, pp. 345-354, 2008

Authors: Piscaer, T.M. | van Osch, G.J.V.M. | Verhaar, J.A.N. | Weinans, H.

Article Type: Research Article

Abstract: Normally, tissue alterations in small animal models for osteoarthritis (OA) are assessed by time-consuming and destructive histology or biochemical assays. Some high resolution imaging modalities are used for longitudinal monitoring of the OA disease process in vivo. μCT is one of these imaging modalities, which is known for superb high-resolution imaging of bone architecture alterations. A major drawback of μCT is that it has low soft-tissue contrast, which makes direct imaging of cartilage impossible. The use of μCT in combination with negatively charged radiopaque contrast agents enables imaging of cartilage degeneration. We demonstrate the possibility of μCT to image cartilage …degeneration as a consequence of experimental OA, by the use contrast enhanced μCT in vivo in a rat model for OA. Furthermore, for the assessment of alterations in molecular processes involved in OA we used the recently developed technique of multi pinhole SPECT. This enables us to assess molecular processes involved in experimental OA in a rat at sub-millimeter level. Here we show quantification of subchondral bone turnover in an OA rat knee. These new techniques demonstrate the possibilities of quantitative experimental OA assessment in small animal models such as mice and rats and might enable substitution of the conventional destructive methods. Show more

Keywords: Osteoarthritis, micro-CT (μCT), imaging, small animal, cartilage, SPECT, scintigraphy, rat, mouse, rabbit, Guinea pig, contrast, ioxaglate

DOI: 10.3233/BIR-2008-0482

Citation: Biorheology, vol. 45, no. 3-4, pp. 355-364, 2008

Authors: Summers, Garry C. | Merrill, Alison | Sharif, Mohammed | Adams, Michael A.

Article Type: Research Article

Abstract: Articular cartilage swells when its collagen network is degraded, both in osteoarthritis (OA) and following mechanical trauma. However, most of the experimental evidence actually shows that it is small excised samples of cartilage that swell, implying that the cartilage was not greatly swollen in-situ before it was excised. We hypothesise that degraded cartilage can be prevented from swelling in-situ by restraint from adjacent normal cartilage and subchondral bone. Four adjacent osteochondral specimens, 20×20 mm, were obtained from regions of the humeral heads of each of 11 skeletally-mature cows. The central region of each specimen was injured by compressive loading …using a 9 mm-diameter flat metal indenter, and cartilage surface damage was confirmed using Indian ink. Damaged cartilage was allowed to swell in physiological saline for 1 h under one of four conditions of restraint: (A) normal in-situ restraint from subchondral bone and surrounding cartilage, (B) restraint from bone only, (C) restraint from cartilage only, (D) no restraint (excised specimen). Cartilage hydration was assessed by freeze-drying to constant weight. Proteoglycan loss from damaged cartilage was quantified by analyzing the GAG content of the surrounding bath using the DMB assay. Hydration of damaged cartilage after swelling depended on restraint (p<0.001), averaging: (A) 76.8%, (B) 78.2%, (C) 78.0%, (D) 81.3%. GAG loss following cartilage surface damage was insufficient to explain observed differences in hydration. The 6% increase in hydration between (A) and (D) can be attributed to swelling which is prohibited when the cartilage remains in-situ. Swelling of degraded cartilage can be largely prevented if it remains in-situ, supported by adjacent healthy bone and cartilage. Adverse physico-chemical consequences of cartilage degradation and swelling may become apparent only when this support is diminished, either because the affected region is large, or following deterioration of adjacent bone or cartilage. Show more

Keywords: Articular cartilage, swelling, restrain, damage, in vitro

DOI: 10.3233/BIR-2008-0495

Citation: Biorheology, vol. 45, no. 3-4, pp. 365-374, 2008

Authors: Werkmeister, E. | de Isla, N. | Marchal, L. | Stoltz, J.F. | Dumas, D.

Article Type: Research Article

Abstract: In articular hyaline cartilage, chondrocytes are surrounded by an extracellular matrix which is mainly composed by collagen and proteoglycanes. Pathological specimens show a partial or complete degradation of this matrix. Therefore, it could be interesting to know how mechanical or biochemical constraints applied to cartilage specimens induce modifications of the cartilage network. Multiphoton technology combined to Second Harmonic Generation (SHG) enables to image cartilage specimens in a non-invasive mode with high resolution at deep penetration. By placing a band pass filter in front of the transmitted light detector, SHG signal with frequency doubled can be isolated for a new …contrast imaging. SHG (second harmonic generation) is a diffusion process generated from organized structures and does not need any fluorescent staining. Due to their non-centrosymetric structure, collagen fibrilles present a high second-order non-linear susceptibility and thus give rise to a strong SHG signal when exposed to high enough electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton microscopy). As the extracellular matrix of cartilage is in part constituted by collagen fibers, it can be imaged with this contrast tool. The intensity of SHG signals strongly depends on the organization of collagen fibers. Thus a modification of the extracellular matrix in terms of 3D-organization of collagen induced by mechanical stress can be shown with this contrast tool. Show more

Keywords: Multiphoton microscopy, second harmonic generation, collagen, extracellular matrix

DOI: 10.3233/BIR-2008-0483

Citation: Biorheology, vol. 45, no. 3-4, pp. 375-383, 2008

Authors: Griffin, Timothy M. | Guilak, Farshid

Article Type: Research Article

Abstract: Obesity is one of the most significant, and potentially most preventable, risk factors for the development of osteoarthritis, and numerous studies have shown a strong association between body mass index and osteoarthritis of the hip, knee, foot and hand. However, the mechanism(s) by which obesity contributes to the onset and progression of osteoarthritis are not fully understood. The strong association between body mass index, altered limb alignment, and osteoarthritis of the knee – and the protective effects of weight loss – support the classic hypothesis that the effects of obesity on the joint are due to increased biomechanical loading and …associated alterations in gait. However, obesity is now considered to be a low-grade systemic inflammatory disease, and recent studies suggest that metabolic factors associated with obesity alter systemic levels of pro-inflammatory cytokines that are also associated with osteoarthritis. Thus, the ultimate influence of obesity on osteoarthritis may involve a complex interaction of genetic, metabolic, and biomechanical factors. In this respect, mouse models of obesity can provide excellent systems in which to examine causal relationships among these factors. In recent years, there have been surprisingly few reports examining the effects of obesity on osteoarthritis using mouse models. In this paper, we review studies on mice and other animal models that provide both direct and indirect evidence on the role of obesity and altered diet in the development of osteoarthritis. We also examine the use of different body mass indices for characterizing “obesity” in mice by comparing these indices to typical adiposity levels observed in obese humans. Taken together, evidence from studies using mice suggest that a complex interaction of environmental and genetic factors associated with obesity contribute to the incidence and severity of osteoarthritis. The ability to control these factors, together with the development of methods to conduct more intricate measures of local biomechanical factors, make mouse models an excellent system to study obesity and osteoarthritis. Show more

Keywords: Inflammation, adiposity, animal models, high fat diet, body mass index, guinea pig, adipokine, leptin

DOI: 10.3233/BIR-2008-0485

Citation: Biorheology, vol. 45, no. 3-4, pp. 387-398, 2008

Authors: Ray, Alpana | Ray, Bimal K.

Article Type: Research Article

Abstract: A number of risk factors including biomechanical stress on the articular cartilage imposed by joint overloading due to obesity, repetitive damage of the joint tissues by injury of the menisci and ligaments, and abnormal joint alignment play a significant role in the onset of osteoarthritis (OA). Genetic predisposition can also lead to the formation of defective cartilage matrix because of abnormal gene expression in the cartilage-specific cells. Another important biochemical event in OA is the consequence of inflammation. It has been shown that synovial inflammation triggers the synthesis of biological stimuli such as cytokines and growth factors which subsequently reach …the chondrocyte cells of the articular cartilage activating inflammatory events in the chondrocytes leading to cartilage destruction. In addition to cartilage degradation, hypertrophy of the subchondral bone and osteophyte formation at the joint margins also takes place in OA. Both processes involve abnormal expression of a number of genes including matrix metalloproteinases (MMPs) for cartilage degradation and those associated with bone formation during osteophyte development. To address how diverse groups of genes are activated in OA chondrocyte, we have studied their induction mechanism. We present evidence for abundant expression of an inflammation-responsive transcription factor, SAF-1, in moderate to severely damaged OA cartilage tissues. In contrast, cells in normal cartilage matrix contain very low level of SAF-1 protein. SAF-1 is identified as a major regulator of increased synthesis of MMP-1 and -9 and pro-angiogenic factor, vascular endothelial growth factor (VEGF). While VEGF by stimulating angiogenesis plays a key role in new bone formation in osteophyte, increase of MMP-1 and -9 is instrumental for cartilage erosion in the pathogenesis of OA. Increased expression in degenerated cartilage matrix and in the osteophytes indicate for a key regulatory role of SAF-1 in directing catabolic matrix degrading and anabolic matrix regenerating activities. Show more

Keywords: SAF-1, MMP-1, MMP-9, VEGF, gene expression, angiogenesis, cartilage degeneration, osteophyte

DOI: 10.3233/BIR-2008-0500

Citation: Biorheology, vol. 45, no. 3-4, pp. 399-409, 2008

Authors: Iannone, Florenzo | Lapadula, Giovanni

Article Type: Research Article

Keywords: Integrins, fibronectin, neuropeptides, substance-P

DOI: 10.3233/BIR-2008-0504

Citation: Biorheology, vol. 45, no. 3-4, pp. 411-413, 2008

Authors: Kirchmeyer, M. | Deffaud, J. | Sebillaud, S. | Moulin, D. | Koufany, M. | Netter, P. | Bianchi, A. | Jouzeau, J.-Y.

Article Type: Research Article

Abstract: In inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-α or -β were expressed at the mRNA level in both species, although IL-1β (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in …both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation. Show more

Keywords: Rheumatoid arthritis, all-trans retinoic acid, metalloproteases, AP-1

DOI: 10.3233/BIR-2008-0486

Citation: Biorheology, vol. 45, no. 3-4, pp. 415-432, 2008

Authors: de Isla, N.G. | Stoltz, J.F.

Article Type: Research Article

Abstract: Osteoarthritis (OA) is a progressive joint disease which represents a combination of several disorders leading to cartilage degradation. The main characteristic of OA is an imbalance between chondrocyte anabolic and catabolic activities. Cytokines produced by the synovium and chondrocytes, especially interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α), play a significant role in the degradation of cartilage. They stimulate the production of nitric oxide (NO), which is involved in cartilage catabolism and also may induce the apoptosis of chondrocytes. The IL-1β produced in activated chondrocytes or synovium may modulate disease progression in OA and should therefore be considered a …potential target for therapeutic interventions. Drug and non-drug treatments are used to relieve pain and/or swelling in OA. Diacerein is a slow-acting drug that may slow down the breakdown of cartilage and relieve pain and swelling. It is not clear whether diacerein works but it has been proposed that diacerein acts as a symptom-modifying and perhaps disease-structure modifying drug. Show more

Keywords: Cartilage, osteoarthritis, diacerein, interleukin-1

DOI: 10.3233/BIR-2008-0503

Citation: Biorheology, vol. 45, no. 3-4, pp. 433-438, 2008

Authors: Deffaud, J. | Kirchmeyer, M. | Domagala, F. | Ficheux, H. | Netter, P. | Bianchi, A. | Jouzeau, J.-Y.

Article Type: Research Article

Abstract: The present work aimed to take advantage of the screening capacity of protein arrays to search for additional targets of rhein in interleukin (IL)-1-stimulated chondrocytes. Primary cultures of chondrocytes from osteoarthritic (OA) patients were stimulated for 24 and 48 h with 1 ng/ml of IL-1α, in the presence or absence of 10−5 M of rhein. Culture supernatants were analyzed with arrays membranes consisting of 120 antibodies directed against cytokines, chemokines, and angiogenic or growth factors and were controlled for 8 proteins by specific immuno-enzymatic assays (ELISA). Protein arrays showed that several CC or CXC chemokines, the growth factor GM-CSF, …the cytokines IL-6, IL-7 and IL-10 (but unexpectedly not IL-1β or TNFα) and the adhesion molecule ICAM-1 were induced maximally by IL-1α. In IL-1-stimulated chondrocytes, rhein reduced slightly the production of MCP-1 and increased those of IL-1Ra, of the cytokine receptors sgp130, IL-6R, sTNFR I and R II, but also of some chemokines or ICAM-1. Specific ELISAs confirmed the effect of rhein on MCP-1, IL-1Ra, sgp130, IL-6R and sTNFR II but was discrepant for GROα and were always more sensitive than protein arrays to detect IL-1 effects such as IL-1Ra and TNFα release. The present data show that rhein modulated some IL-1-induced responses contributing possibly to its chondroprotective (IL-1Ra, MCP-1) or cytokine modifying (sTNFR II, sgp130) properties, but that protein arrays were poorly sensitive to check for IL-1- and/or rhein-induced changes. Show more

Keywords: Rhein, osteoarthritis, cartilage, protein screening, pro-inflammatory cytokines, chemokines

DOI: 10.3233/BIR-2008-0484

Citation: Biorheology, vol. 45, no. 3-4, pp. 439-455, 2008

Authors: De Croos, J.N.A. | Pilliar, R.M. | Kandel, R.A.

Article Type: Research Article

Abstract: Generating bioengineered cartilage yields tissue with physical qualities inferior to that of native tissue. Application of cyclic compression (30 min, 1 kPa, 1 Hz) to cartilage cells (chondrocytes) seeded on calcium polyphosphate substrates significantly increases the accumulation of collagens and proteoglycans by 24 hours, thus improving the tissue generated. The mechanism for this increase is not fully known but seems to follow a remodeling pathway of sequential catabolic and anabolic changes. The initial catabolic event involves increased transcription of matrix metalloproteinase (MMP)-3 and MMP-13 two hours after the end of cyclic compression. As MMP-3 and MMP-13 promoters contain activating protein-1 …(AP-1) DNA binding sites, we investigated the effect of inhibiting DNA binding through the use of modified decoy oligodeoxynucleotides (ODN). Mechanical stimulation in the presence of the ODN blocked AP-1 DNA binding as detected by electrophoretic mobility shift assay and prevented the increased transcription of MMP-3 and MMP-13. As well the increased accumulation of collagens and proteoglycans by 24 hours in mechanically stimulated samples was prevented. The data suggests that the mechano-induction of MMP-3 and MMP-13 may be regulated at the AP-1 DNA binding site and that upregulation of these metalloproteases is a necessary component of the matrix remodeling initiated by cyclic compression. Show more

Keywords: Mechanical stimulation, decoy oligodeoxynucleotides, chondrocytes, tissue engineering

DOI: 10.3233/BIR-2008-0488

Citation: Biorheology, vol. 45, no. 3-4, pp. 459-469, 2008

Authors: Raimondi, Manuela T. | Candiani, Gabriele | Cabras, Mariasara | Cioffi, Margherita | Laganà, Katia | Moretti, Matteo | Pietrabissa, Riccardo

Article Type: Research Article

Abstract: We have studied an in vitro engineered cartilage model, consisting of bovine articular chondrocytes seeded on micro-porous scaffolds and perfused with very low regimens of interstitial flow. Our previous findings suggested that synthesis of sulphated glycosaminoglycans (sGAG) was promoted in this model, if the level of shear generated on cells was maintained below 10 mPa (0.1 dyn/cm2 ). Constructs were stimulated with a median shear stress of 1.2 and 6.7 mPa using two independent culture chambers. Quantification of the applied stresses and of oxygen consumption rates was obtained from computational modelling. Experimentally, we set a time zero reference at 24 …hours after cell seeding and total culture time at two weeks. The cell metabolic activity, measured by MTT, was significantly lower in all constructs at two weeks (−73% in static controls, −66% in the 1.2 mPa group and −60% in the 6.7 mPa group) vs. the time zero group, and significantly higher (+33%) in the 7 mPa group vs. static controls. The ratio between synthesis of collagen type II/type I, measured by Western Blot, was significantly higher in the 1.2 mPa constructs (+109% vs. the 6.7 mPa group, +120% vs. the time zero group and +286% vs. static controls). A trend of decreased α-actin expression was observed with increased ratio of type II to type I collagen, in all groups. These results reinforce the notion that, at early time points in culture, hydrodynamic shear below 10 mPa may promote formation of extra-cellular matrix specific to hyaline cartilage in chondrocyte-seeded constructs. Show more

Keywords: Tissue engineering, cartilage, mechanobiology, shear, oxygen, perfusion, porous scaffold

DOI: 10.3233/BIR-2008-0490

Citation: Biorheology, vol. 45, no. 3-4, pp. 471-478, 2008

Authors: Elder, Steven H. | Shim, Joon Wan | Borazjani, Ali | Robertson, Hess M. | Smith, Kathryn E. | Warnock, James N.

Article Type: Research Article

Abstract: Undifferentiated connective tissue that arises during embryonic development and some healing processes contains pluripotent mesenchymal stem cells. It is becoming increasingly evident that the mechanical environment is an important differentiation factor for these cells. In our laboratory, we have focused on the potential for mechanical signals to induce chondrogenic differentiation of mesenchymal stem cells. Using C3H10T1/2 cells as a model, we have investigated the influence of hydrostatic pressure, equibiaxial contraction, and centrifugal pressure on chondroinduction. Cells responded to cyclic hydrostatic compression (5 MPa at 1 Hz) and cyclic contractile strain (15% at 1 Hz) by upregulating aggrecan and collagen type …II gene expression. In addition, a preliminary study of the effects of centrifugal pressure (4.1 MPa for 30 min) suggests that it may increase cell proliferation and stimulate proteoglycan and collagen type II production. We speculate that compression, whether it is distortional or hydrostatic in nature, applied to undifferentiated connective tissue triggers differentiation toward a chondrocyte-like phenotype and production of a less permeable extracellular matrix which is capable of sustaining increasingly higher hydrostatic fluid pressure for compressive load support. Show more

Keywords: Mesenchymal stem cells, chondrogenic differentiation, hydrostatic pressure, equibiaxial contraction, centrifugal pressure

DOI: 10.3233/BIR-2008-0496

Citation: Biorheology, vol. 45, no. 3-4, pp. 479-486, 2008

Authors: McNulty, Amy L. | Guilak, Farshid

Article Type: Research Article

Abstract: Current therapies for meniscal injury seek to preserve and repair damaged tissue since loss of meniscal tissue is associated with degenerative changes in the joint, ultimately leading to osteoarthritis (OA). After a meniscal tear, the difficulty of integrating juxtaposed meniscal surfaces continues to be an obstacle. In order to determine the local factors that are necessary for successful tissue repair, previous studies have developed in vitro model systems that allow both biological and quantitative biomechanical measures of meniscus repair. Many studies have shown the importance of individual factors in meniscus metabolism, but there is a complex interplay among a variety …of factors that influence meniscal healing, including inflammatory cytokines, growth factors, mechanical loading, and zonal differences in cell and tissue properties. In particular, the upregulation of inflammatory cytokines following joint injury appears to have significant catabolic influences on meniscal cell metabolic activity that must be overcome in order to promote repair. In the presence of inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor alpha (TNF-α), intrinsic meniscal repair in vitro is significantly inhibited. While anabolic growth factors, such as transforming growth factor-β1 (TGF-β1), enhance meniscal repair, they cannot completely overcome the IL-1-mediated inhibition of repair. The mechanisms by which these mediators influence meniscal repair, and their interactions with other factors in the microenvironment, such as mechanical loading, remain to be determined. Future studies must address these complex interactions during meniscal healing to ultimately enhance meniscal repair. Show more

Keywords: Articular cartilage, matrix metalloproteinases, fibrochondrocyte, cell migration, meniscectomy, mechanical compression

DOI: 10.3233/BIR-2008-0489

Citation: Biorheology, vol. 45, no. 3-4, pp. 487-500, 2008

Authors: Moutos, Franklin T. | Guilak, Farshid

Article Type: Research Article

Abstract: Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types …of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds. Show more

Keywords: Articular cartilage, tissue engineering, stem cell, hydrogel, fiber, biomaterial

DOI: 10.3233/BIR-2008-0491

Citation: Biorheology, vol. 45, no. 3-4, pp. 501-512, 2008

Authors: Cournil-Henrionnet, Christel | Huselstein, Céline | Wang, Yun | Galois, Laurent | Mainard, Didier | Decot, Véronique | Netter, Patrick | Stoltz, Jean-François | Muller, Sylvaine | Gillet, Pierre | Watrin-Pinzano, Astrid

Article Type: Research Article

Abstract: Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, …and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0–P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes. Show more

Keywords: Human mesenchymal stem cells, bone marrow, chondrocytes, cartilage engineering, gene expression, flow cytometry

DOI: 10.3233/BIR-2008-0487

Citation: Biorheology, vol. 45, no. 3-4, pp. 513-526, 2008

Authors: Wang, Y. | De Isla, N. | Decot, V. | Marchal, L. | Cauchois, G. | Huselstein, C. | Muller, S. | Wang, B.H. | Netter, P. | Stoltz, J.F.

Article Type: Research Article

Abstract: To investigate whether the chondrocytes–alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3×106 cells/ml) or high density (HD: 10×106 cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M …phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell–cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage. Show more

Keywords: Cell seeding density, alginate, redifferentiation, chondrocytes, tissue engineering

DOI: 10.3233/BIR-2008-0493

Citation: Biorheology, vol. 45, no. 3-4, pp. 527-538, 2008

Authors: Tiitu, V. | Pulkkinen, H.J. | Valonen, P. | Pulliainen, O. | Kellomäki, M. | Lammi, M.J. | Kiviranta, I.

Article Type: Research Article

Abstract: In the present study bovine chondrocytes were cultured in two different environments (static flasks and bioreactor) in knitted poly-L,D-lactide (PLDLA) scaffolds up to 4 weeks. Chondrocyte viability was assessed by employing cell viability fluorescence markers. The cells were visualized using confocal laser scanning microscopy and scanning electron microscopy. The mechanical properties and uronic acid contents of the scaffolds were tested. Our results showed that cultivation in a bioreactor improved the growth and viability of the chondrocytes in the PLDLA scaffolds. Cells were observed both on and in between the fibrils of scaffold. Furthermore, chondrocytes cultured in the bioreactor, regained …their original round phenotypes, whereas those in the static flask culture were flattened in shape. Confocal microscopy revealed that chondrocytes from the bioreactor were attached on both sides of the scaffold and sustained viability better during the culture period. Uronic acid contents of the scaffolds, cultured in bioreactor, were significantly higher than in those cultured in static flasks for 4 weeks. In summary, our data suggests that the bioreactor is superior over the static flask culture when culturing chondrocytes in knitted PLDLA scaffold. Show more

Keywords: Biomaterials, bioreactor, chondrocyte, polylactic acid, tissue engineering

DOI: 10.3233/BIR-2008-0492

Citation: Biorheology, vol. 45, no. 3-4, pp. 539-546, 2008