Search

Interstitial fluid load support (FLS) is a dominant mechanism of lubrication in cartilage, producing a low friction coefficient while enhancing the tissue's load bearing capabilities. Due to its viscosity, synovial fluid (SF) may retard loss of FLS by slowing the exudation of interstitial fluid from the cartilage. This study tested this hypothesis by comparing the stress-relaxation (SRL) response of immature bovine articular cartilage immersed either in phosphate buffered saline (PBS) or in healthy mature bovine SF, under unconfined compression (fluid exudation across cut lateral tissue boundary) and indentation testing (fluid exudation across articular surface). To investigate the influence of diffusion of SF molecular constituents into cartilage, the effect of incubation time in SF on SRL was also investigated. The SRL response in unconfined compression was not significantly different in PBS versus SF when compared directly (p = 0.98) and had a slope ofm = 1.00 ± 0.04 (R 2  = 0.989 ± 0.007). Samples tested in PBS exhibited characteristic relaxation times, τ PBS =42.6 ± 5.3 s andτ SF  = 40.8 ± 4.7 s, that were not significantly different (p = 0.40). Incubation time of 24 h in SF resulted in no significant difference in the SRL response (p = 0.39, m=1.03 ± 0.12; R 2 =0.983 ± 0.011, andτ PBS  = 43.4 ± 10.7 s versusτ SF  = 41.5 ± 4.8 s, p = 0.59). Indentation testing showed some statistically significant, but functionally insignificant, difference in SRL responses in PBS versus SF with a slope ofm = 0.958 ± 0.060 (R 2  = 0.957 ± 0.020, p = 0.029, andτ PBS  = 16.9 ± 2.6 s versusτ SF  = 19.4 ± 3.3 s, p = 0.073). Based on these results, we reject the hypothesis that healthy SF can retard the loss of FLS in cartilage due to its viscosity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Allografts of articular cartilage are both used clinically for tissue-transplantation procedures and experimentally as model systems to study the physiological behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Chemical mediators in poorly defined culture media can stimulate cells to quickly degrade their surrounding extracellular matrix. This is particularly true of juvenile cartilage which is generally more responsive to chemical stimuli than mature tissue. By carefully modulating the culture media, however, it may be possible to preserve allograft tissue over the long-term while maintaining its original mechanical and biochemical properties. In this study juvenile bovine cartilage explants (both chondral and osteochondral) were cultured in both chemically defined medium and serum-supplemented medium for up to 6 weeks. The mechanical properties and biochemical content of explants cultured in chemically defined medium were enhanced after 2 weeks in culture and thereafter remained stable with no loss of cell viability. In contrast, the mechanical properties of explants in serum-supplemented medium were degraded by ( approximately 70%) along with a concurrent loss of biochemical content (30-40% GAG). These results suggest that long-term maintenance of allografts can be extended significantly by the use of a chemically defined medium.

An accurate description of the mechanical environment around chondrocytes embedded within their dense extracellular matrix (ECM) is essential for the study of mechano-signal transduction mechanism(s) in explant experiments. New methods have been developed to determine the inhomogeneous strain distribution throughout the depth of the ECM during compression (Schinagl et al., 1996, Annals of Biomedical Engineering 24, 500-512; Schinagl et al 1997. Journal of Orthopaedics Research 15, 499-506) and the corresponding depth-dependent aggregate modulus distribution (Wang and Mow, 1998. Transactions of the Orthopaedics Research Society 23, 484; Chen and Sah, 1999. Transactions of the Orthopaedics Research Society 24, 635). These results provide the motivation for the current investigation to assess the influence of tissue inhomogeneity on the chondrocyte milieu in situ, e.g. stress, strain, fluid velocity and pressure fields within articular cartilage. To describe this inhomogeneity, we adopted the finite deformation biphasic constitutive law developed by Holmes and Mow (1990 Journal of Biomechanics 23, 1145-1156). Our calculations show that the mechanical environment inside an inhomogeneous tissue differs significantly from that inside a homogeneous tissue. Furthermore, our results indicate that the need to incorporate an inhomogeneous aggregate modulus. or an anisotropy, into the biphasic theory to describe articular cartilage depends largely on the motivation for the study.

We investigated the effect of newborn bovine serum on the intracellular calcium [Ca(2+)](i) response of primary cultured bone cells stimulated by fluid flow. As it has been previously established that these cells exhibit [Ca(2+)](i) responses to fluid flow shear stress in saline media without growth factors or other chemically stimulatory factors, we hypothesized that the addition of serum to the flow medium would enhance the mechanosensitivity of the cells. We examined the effect of a short-term (10-15min) exposure of the cells to 2 and 10% serum prior to flow stimulation (pretreated) compared to not exposing the cells prior to flow stimulation (unpretreated). The cells were subjected to a well-defined, 90-s flow stimulus with shear stress levels ranging from 0.02 to 3.5Pa in a laminar flow chamber using a saline medium supplemented with 2 or 10% serum. For pretreatment, the serum concentration was the same from pre-flow to flow exposure. We observed a differential effect in the magnitude of the peak [Ca(2+)](i) response modulated by the concentration of serum in the pre-flow medium. Additionally, ATP-supplemented flow was examined as a comparison to the serum-supplemented flow and exhibited a similar trend in the peak [Ca(2+)](i) flow response that was dependent on ATP concentration and pre-flow exposure conditions. These findings demonstrate that under the conditions of this study, chemical agonist exposure can modulate the [Ca(2+)](i) response in bone cells subjected to fluid flow-induced shear stress.

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.

In this study, we sought to determine if there is a requirement for calcium entry from the extracellular space as well as calcium from intracellular stores to produce real-time intracellular calcium responses in cultured bone cells subjected to fluid flow. Understanding calcium cell signaling may help to elucidate the biophysical transduction mechanism(s) mediating the conversion of fluid flow to a cellular signal. An experimental design which utilized a scheme of pharmacological blockers was employed to distinguish between the biochemical pathways involved in this cell signaling. A parallel-plate flow chamber served as the cell stimulating apparatus and a fluorescence microscopy system using the calcium-sensitive dye fura-2 measured the intracellular calcium changes. In the present study, evidence for a role by the inositol-phospholipid biochemical pathway, specifically inositol trisphosphate (IP3) was obtained using neomycin which completely inhibited the calcium response to flow. Additionally, a concomitant role of extracellular calcium was demonstrated through experiments performed in calcium-free medium which also eliminated the flow response. Experiments conducted with gadolinium, a stretch-activated channel blocker, partially inhibited (approximately 30%) the flow response while verapamil, a type-L voltage sensitive channel blocker, had no effect on the flow response. These results suggest a requirement of extracellular calcium (or calcium influx) as well as IP3-induced calcium release from intracellular stores for generating the intracellular calcium response to flow in bone cells.

Cultured cells subjected to fluid flow are exposed to mechanical forces and electrokinetic forces. The convective current establishes an electrokinetic force created by the flow-dependent transport of mobile ions in the media over the charged cell surfaces. This current can be expressed as a current density, the current normalized by the cross-sectional area in which it exists. In this study, we hypothesized that the convective current density has no role in the bone cell real-time intracellular calcium response to fluid flow. Our hypothesis was tested by incorporating electrokinetic measurements and classical electrokinetic double-layer theory to estimate the value of convective current density in a parallel-plate flow chamber and then to apply an external current during the presence of fluid flow that would alter convective current density. There was no difference between the mean peak calcium response of cells exposed to flow with an altered (canceled or doubled) convective current density versus flow with an unmodified convective current density, as was measured with fura-2 fluorescence microscopy. These results suggest that mechanical forces, such as fluid-induced shear stress, rather than concomitant electrokinetic forces are the primary stimuli in eliciting the observed calcium response of bone cells to fluid flow.

A device is described which is designed to bring about uniform and equi-biaxial strains of 0.04-0.4% on a circular elastomeric sheet upon which cells are grown in a monolayer. Strain measurements at six different radii of the well provide evidence of the uniformity of the strain field over the 2.54 cm diameter of the well with a mean strain of 0.15% and a standard deviation of 0.01% strain. Major advantages of the system over those previously described are that (1) the substrate remains in the same plane throughout the application of stretch, (2) the device is designed to provide a state of uniform equi-biaxial strain to the entire substrate of the cells and (3) all cells 'see' the same strain field, no matter what their orientation. This allows for real-time optical monitoring of the cells throughout the stretching cycle of the cell substrate. In this way, changes in the cell membrane, cell nucleus, cytoskeleton and in fluorescently labelled calcium can be monitored microscopically during application of an equi-biaxial mechanical stretch.

A serum assay method incorporating two internal standards suitable for routine clinical analysis of tricyclic antidepressant drugs at the therapeutic level is described. The method involves extraction and chromatography using a reversed-phase high-performance liquid chromatography system incorporating pairing and organic counter-ions. The quantitative characteristics of the method are reported and results are compared with consensus values for four commonly encountered drugs, amitriptyline, nortriptyline, imipramine and desipramine. The method is shown to be applicable to more recently developed antidepressants such as nomifensine and maprotiline and the flexibility of the chromatography is discussed. Sample chromatograms obtained from patient samples in the presence and absence of potentially interfering drugs and metabolites are discussed.

An ion-pair reversed-phase high-performance liquid chromatographic method is presented for the determination of adriamycin and adriamycinol concentrations in rat serum and tissues. The scheme for developing the chromatographic conditions is discussed. A good separation of adriamycinol and adriamycin was obtained by using a high sodium lauryl sulphate concentration in the mobile phase. The retention times of adriamycinol, adriamycin and daunomycin (internal standard) were less than 3 min with no interfering peaks from endogenous constituents. A protein precipitation method was used to prepare the serum and tissue samples for injection.