A biphasic multiscale study of the mechanical microenvironment of chondrocytes within articular cartilage under unconfined compression

Computational analyses have been used to study the biomechanical microenvironment of the chondrocyte that cannot be assessed by in vitro experimental studies; yet all computational studies thus far have focused on the effect of zonal location (superficial, middle, and deep) on the mechanical microenvironment of chondrocytes. The aim of this paper was to study the effect of both zonal and radial locations on the biomechanical microenvironment of chondrocytes in inhomogeneous cartilage under unconfined stress relaxation. A biphasic multiscale approach was employed and nine chondrocytes in different locations were studied. Hyperelastic biphasic theory and depth-dependent aggregate modulus and permeability of articular cartilage were included in the models. It was found that both zonal and radial locations affected the biomechanical stresses and strains of the chondrocytes. Chondrocytes in the mid-radial location had increased volume during the early stage of the loading process. Maximum principal shear stress at the interface between the chondrocyte and the extracellular matrix (ECM) increased with depth, yet that at the ECM–pericellular matrix (PCM) interface had an inverse trend. Fluid pressure decreased with depth, while the fluid pressure difference between the top and bottom boundaries of the microscale model increased with depth. Regardless of location, fluid was exchanged between the chondrocyte, PCM, and ECM. These findings suggested that even under simple compressive loading conditions, the biomechanical microenvironment of the chondrocytes, PCM and ECM was spatially dependent. The current study provides new insight on chondrocyte biomechanics.     

Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading

Articular cartilage in vivo experiences the effects of both cell-regulatory proteins and mechanical forces. This study has addressed the hypothesis that the frequency of intermittently or continuously applied mechanical loads is a critical parameter in the regulation of chondrocyte collagen biosynthesis. Cyclic compressive pressure was applied intermittently to bovine articular cartilage explants by using a sinusoidal waveform of 0.1–1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5–20 s followed by a load-free period of 10–1,000 s. These loading protocols were repeated for a total duration of 6 days. In separate experiments, cyclic loading was continuously applied by using a sinusoidal waveform of 0.001–0.5 Hz frequency and a peak stress of 1.0 MPa for a period of 3 days. Unloaded cartilage discs of the same condyle were cultured in identically constructed loading chambers and served as controls. We report quantitative data showing that (1) no correlation exists between the relative rate of collagen synthesis expressed as the proportion of newly synthesized collagen among newly made proteins and either the frequency of intermittently or continuously applied loads or the overall time cartilage is actively loaded, and (2) individual protocols of intermittently applied loads can reduce the relative rate of collagen synthesis and increase the water content, whereas (3) continuously applied cyclic loads always suppress the relative rate of collagen synthesis compared with that of unloaded control specimens. The results provide further experimental evidence that collagen metabolism is difficult to manipulate by mechanical stimuli. This is physiologically important for the maintainance of the material properties of collagen in view of the heavy mechanical demands made upon it. Moreover, the unaltered or reduced collagen synthesis of cartilage explants might reflect more closely the metabolism of normal or early human osteoarthritic cartilage.This work was supported by the Federal Ministry of Education and Research (BMBF no. 0311058) and by the foundation S.E.T.     

Inhibition of interleukin-1-induced synovitis and articular cartilage proteoglycan loss in the rabbit knee by recombinant human interleukin-1 receptor antagonist

Intra-articular injection of interleukin-1 (IL-1) into the knee joints of rabbits produces a synovitis associated with the loss of proteoglycan from the matrix of articular cartilage. This experimental finding supports the hypothesis that IL-1 is a possible mediator of the pathology of inflammatory joint diseases and suggests that antagonism of IL-1 could offer a therapeutic approach to these diseases. It has recently been reported that culture of human monocytes on adherent IgG stimulates these cells to synthesize a specific inhibitor of IL-1 bioactivity (IL-1ra) that acts as a receptor antagonist with lymphocytes and mesenchymal cells. We have now shown that intravenous injection of IL-1ra into rabbits given an intra-articular injection of recombinant IL-1 beta not only inhibits the entry of leukocytes into the synovial lining and joint cavity but blocks the ability of IL-1 to cause loss of proteoglycan from articular cartilage. This ability of IL-1ra to inhibit IL-1-induced arthritis in the rabbit reveals that this protein has appropriate pharmacokinetic and pharmacodynamic properties and further strengthens the belief that it may be a useful therapeutic agent.     

A fibril reinforced nonhomogeneous poroelastic model for articular cartilage: inhomogeneous response in unconfined compression

The depth dependence of material properties of articular cartilage, known as the zonal differences, is incorporated into a nonlinear fibril-reinforced poroelastic model developed previously in order to explore the significance of material heterogeneity in the mechanical behavior of cartilage. The material variations proposed are based on extensive observations. The collagen fibrils are modeled as a distinct constituent which reinforces the other two constituents representing proteoglycans and water. The Young's modulus and Poisson's ratio of the drained nonfibrillar matrix are so determined that the aggregate compressive modulus for confined geometry fits the experimental data. Three nonlinear factors are considered, i.e. the effect of finite deformation, the dependence of permeability on dilatation and the fibril stiffening with its tensile strain. Solutions are extracted using a finite element procedure to simulate unconfined compression tests. The features of the model are then demonstrated with an emphasis on the results obtainable only with a nonhomogeneous model, showing reasonable agreement with experiments. The model suggests mechanical behaviors significantly different from those revealed by homogeneous models: not only the depth variations of the strains which are expected by qualitative analyses, but also, for instance, the relaxation-time dependence of the axial strain which is normally not expected in a relaxation test. Therefore, such a nonhomogeneous model is necessary for better understanding of the mechanical behavior of cartilage.     

Differential effects of hyperosmotic challenge on interleukin-1-activated pathways in bovine articular cartilage

Chondrocytes in situ experience fluctuations in extracellular osmolarity resulting from mechanical loading. The objective of this study was to determine whether hyperosmotic stress causes or exacerbates interleukin-1 (IL-1)-mediated effects in bovine articular cartilage. Disks of cartilage cut from the articular surface of calf radiocarpal joints were incubated for 24h in the presence or absence of IL-1 in Dulbecco's modified Eagle's medium adjusted to various osmolalities with sucrose or NaCl. Cyclooxygenase (COX)-2 levels in the cartilage were examined by Western blot. Culture media were assayed for prostaglandin E(2) (PGE(2)), nitrite as an indicator of nitric oxide (NO) production, and sulfated glycosaminoglycan as an indicator of proteoglycan degradation. We report the osmolality-dependent potentiation of COX-2 and PGE(2) production, and the osmolality-dependent inhibition of NO production and proteoglycan degradation in IL-1-activated cartilage. The data demonstrate that osmotic and cytokine signaling interact to differentially modulate IL-1-stimulated effects in calf articular cartilage.     

The sensitivity of synthesis of human cartilage matrix to inhibition by IL-1 suggests a mechanism for the development of osteoarthritis

The damage to articular cartilage, characteristic of arthritic disease, is usually ascribed to increased degradative activity by enzymes or free radicals from locally activated cells. We propose that inhibition of matrix synthesis, and consequential impairment of the natural repair process, may be at least as important in chronic joint disease.     

A critical role of interleukin-1 in preterm labor

Preterm birth (PTB) is a leading cause of neonatal mortality and morbidity worldwide, and represents a heavy economic and social burden. Despite its broad etiology, PTB has been firmly linked to inflammatory processes. Pro-inflammatory cytokines are produced in gestational tissues in response to stressors and can prematurely induce uterine activation, which precedes the onset of preterm labor. Of all cytokines implicated, interleukin (IL)-1 has been largely studied, revealing a central role in preterm labor. However, currently approved IL-1-targeting therapies have failed to show expected efficacy in pre-clinical studies of preterm labor. Herein, we (a) summarize animal and human studies in which IL-1 or IL-1-targeting therapeutics are implicated with preterm labor, (b) focus on novel IL-1-targeting therapies and diagnostic tests, and (c) develop the case for commercialization and translation means to hasten their development.     

Changes in proteoglycan synthesis of chondrocytes in articular cartilage are associated with the time-dependent changes in their mechanical environment

Explant loading experiments were conducted to investigate the effect of load duration on proteoglycan synthesis. A compressive load of 0.1 MPa applied for 10 min was found to stimulate proteoglycan synthesis, while the same load applied for 20 h suppressed synthesis. This bimodal response suggests that the cells are responding to different mechanical stimuli as time progresses. A theoretical model has therefore been developed to describe the mechanical environment perceived by cells within soft hydrated tissues (e.g. articular cartilage) while the tissue is being loaded. The cells are modeled, using the biphasic theory, as fluid-solid inclusions embedded in and attached to a biphasic extracellular matrix of distinct material properties. A method of solution is developed which is valid for any axisymmetric loading configuration, provided that the cell radius, a, is small relative to the tissue height, h (i.e. h/a 1). A closed-form analytical solution for this inclusion problem is then presented for the confined compression configuration. Results from this model show that the mechanical environment in and around the cells is time dependent and inhomogeneous, and can be significantly influenced by differences in properties between the cell and the extracellular matrix.     

A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.     

Correlation of neutrophil and monocyte derived interleukin-1 receptor antagonist and interleukin-8 with colitis severity in the rabbit

Activated neutrophils and monocytes produce interleukin (IL)-8, a pro-inflammatory chemokine, but also IL-1 receptor antagonist (IL-1ra), which is an anti-inflammatory cytokine. We were interested to see the profiles of IL-8 and IL-1ra in the colonic tissue and in the peripheral blood leukocytes (PBL) during the development of immune complex induced colitis in rabbits. IL-1ra and IL-8 in PBL were measured in 26 rabbits at time 0 h, 24 h, and 48 h after induction of colitis. The colons were removed at 48 h for measuring myeloperoxidase (MPO), ulcer area, IL-1ra and IL-8. Epithelial damage, crypt abscess formation and leukocyte infiltration of the colonic tissue were major features of this colitis model. During the development of colitis, there was an increase in circulating neutrophils and monocytes (P < 0.0001), but not lymphocytes. Likewise, elevated amounts of IL-1ra (P = 0.0001) and IL-8 (P = 0.0219) production by PBL were observed following induction of colitis. Flow cytometry revealed major source of IL-1ra was monocytes, while the main sources of IL-8 were neutrophils and monocytes. There was correlation between MPO and ulcer area (R_s = 0.6327, P < 0.0001). At 24 h, PBL from MPO^High group (n = 11) showed increased IL-1ra (P = 0.027) and IL-8 (P = 0.0128) levels vs MPO^Low group (n = 15). IL-8 production by PBL showed correlation with tissue MPO (R_s = 0.4273, P = 0.0295). The colitis in this model was associated with an increase in circulating monocytes and neutrophils, which released increased amounts of IL-8 and IL-1ra. Further, IL-8 and IL-1ra showed correlation with the severity of colitis. These observations should significantly further understandings on the role of neutrophils and monocytes in the immunopathogenesis of ulcerative colitis.     

Monotonicity of interleukin-1 receptor-ligand binding with respect to antagonist in the presence of decoy receptor

We consider the interaction between interleukin-1 IL-1, its receptor IL-1RI, the receptor antagonist IL-1Ra and a decoy receptor (or trap) that binds both with the ligand and the antagonist. We study how the interaction between IL-1Ra and the decoy receptor influences the effect of either reagent on reducing the equilibrium concentration of the receptor-ligand complex. We obtain that, given a certain relationship among the equilibrium constants and the total concentrations of solutes, IL-1Ra can reverse the effect of the decoy receptor of decreasing the equilibrium concentration of the receptor-ligand complex. This finding derives from a mathematical result applicable to any reversible chemical reaction system comprising four species arranged in a square such that each species binds its two immediate neighbors. The result gives the monotonicity of the equilibrium concentrations of the complex species as functions of the total concentrations of the simple species.     

Changes in the metabolism of the proteoglycans from sheep articular cartilage in response to mechanical stress

The effect of altered mechanical stress on the metabolism of sheep articular cartilage has been investigated. A simple experimental model involving the immobilisation of a single sheep foreleg was used to study the effect of increased or decreased functional demand on the chemical composition of, and the incorporation of labelled acetate into, the proteoglycans of sheep articular cartilage. By immobilisation of one of the sheep forelegs, mechanical stress is removed from that particular joint, while increased stress is placed on the other foreleg. The load distribution about the two hind legs remains essentially the same. After a 4-week immobilisation period there was a significant increase in the hexuronic acid content of the cartilage from the loadbearing ankle joint, and a corresponding decrease in the hexuronic acid content of the non-loadbearing joint cartilage. Hexosamine analyses of the cartilage from each joint showed that the major chemical occurred in the chondroitin sulphate fraction. From analyses of the extracted and isolated proteoglycans from each experimental joint it was evident that there was a significant decrease in the molecular weight of the proteoglycan from the non-loadbearing joint. In vitro studies showed increased incorporation of labelled acetate into the chondroitin sulphate fraction from the loadbearing joint but a corresponding decreased incorporation into the non-loadbearing immobilised joint cartilage. These results suggest that the changes observed in the chemical composition of the cartilage from the loadbearing and non-loadbearing joints may be accounted for in part by changes in the biosynthesis of the cartilage proteoglycan in response to altered functional demand.     

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin

Abstract The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E₂ release was studied in monocytes (Mø). Both IL-1α and IL-1β increased the release of PGE₂ in a concentration-dependent manner, with EC₅₀s of 0.48 nM and 0.12 nM, respectively. Intact Mø were prelabelled with [³H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [³H]phosphatidylinositol-4,5,-bisphosphate to aqueous soluble radioactivity by Mø homogenates. IL-1α (5.8 nM) increased the accumulation of IPs within 1–4 minutes and increases in IP₃ and IP₄ occured before the increase in IP₁₊₂ whereas LPS only increased the IPs level after at least 30 min. IL-1α increased PIC activity in Mø homogenates within 15 min with an EC₅₀ of 0.58 nM and IL-1β (0.1 nM) also increased activity. Neither IL-1α nor IL-1β affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in Mø.     

Differential calcium signaling in dairy cows with specific CXCR1 genotypes potentially related to interleukin-8 receptor functionality

Neutrophil migration and activation are critical components of innate immunity and are mediated by a variety of inflammatory mediators, which include interleukin-8 (IL-8) and epithelial-derived neutrophil activating peptide-78 (ENA-78). Limited knowledge on the expression of receptors for these inflammatory mediators (CXCR1 and CXCR2) in bovine, in addition to the association of a polymorphism (G→C) in position +777 of the CXCR1 gene with impaired neutrophil function, prompted evaluation of CXCR1 and CXCR2 mRNA and protein expression, ligand binding affinity, and intracellular receptor signaling in neutrophils from cows with different CXCR1 genotypes. Initial observations revealed that overall IL-8 receptor numbers appeared to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of SB225002, a CXCR2 inhibitor, CXCR1 affinity was about fivefold lower in cows with a CC genotype and may have resulted in an underestimation of receptor numbers in cows with this genotype. In addition, intracellular calcium ([Ca⁺⁺]_i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not ENA-78. Furthermore, when neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, [Ca⁺⁺]_i release was lower in cows with a CC genotype, suggesting differential CXCR1 signaling among genotypes. These findings offer knowledge of the role that each of these receptors plays in the inflammatory response in the bovine and provide insight into the potential mechanisms that may be affected in neutrophils of cows with different CXCR1 genotypes.     

Maternal microchimerism leads to the presence of interleukin-2 in interleukin-2 knock out mice: implications for the role of interleukin-2 in thymic function

The role of interleukin-2 (IL-2) in thymic development is uncertain. Not surprisingly, IL-2 knockout (KO) mice have been used to address this question. However, as we report here, such mice are chimeric, containing both IL-2 KO cells and IL-2-expressing cells transferred in utero from their heterozygous mothers. These cells produce IL-2 in amounts detectable by conventional means, and their presence in lymphoid tissues confounds efforts to define the true IL-2 KO phenotype. To minimize the amount of IL-2 available to the thymus, we subjected recombinase activating gene-1 KO mice to bone marrow transplantation using IL-2 KO donors, and then followed the reconstitution of the thymus. The thymuses of these mice became increasingly aberrant over time, including abnormalities in both stromal cells and thymocytes. These results demonstrate that IL-2 is critical to several aspects of thymic function, a finding previously obscured by the presence of IL-2 in IL-2 KO mice.     

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT_2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT_2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.     

Evidence for interleukin-1-independent stimulation of interleukin-12 and down-regulation by interleukin-10 in Helicobacter pylori-infected murine dendritic cells deficient in the interleukin-1 receptor

Helicobacter pylori infection is characterized by infiltration of cells of the immune system, including dendritic cells, into the gastric mucosa. During chronic inflammation with Helicobacter pylori infection, a variety of cytokines are secreted into the mucosa, including interleukin-1beta (IL-1beta). The role of IL-1 in H. pylori infection was investigated using bone-marrow-derived dendritic cells from wild-type and IL-1 receptor-deficient (IL-1R-/-) mice. Dendritic cells were incubated with H. pylori at a multiplicity of infection of 10 and 100, and cytokine production evaluated. Helicobacter pylori SS1, H. pylori SD4, and an isogenic cagE mutant of SD4 stimulated IL-12, IL-6, IL-1beta, IL-10, and tumor necrosis factor-alpha at comparable levels in dendritic cells from both wild-type and IL-1R-/- mice. IL-10 production required the higher inoculum, while IL-12 was decreased at this bacterial load. Pretreatment of dendritic cells with an antibody to IL-10 resulted in an increased production of IL-12, confirming the down-regulation of IL-12 by IL-10. cagE was required for maximum stimulation of IL-12 by H. pylori. We speculate that the down-regulation of IL-12 by IL-10 at the higher multiplicity of infection represents the modulation of the host inflammatory response in vivo by H. pylori when the bacterial load is high, allowing for persistent colonization of the gastric mucosa.