Biorheology - Volume 45, issue 3-4

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