Biomedical subjects

Alan J Grodzinsky

Publications and source records attributed to Alan J Grodzinsky.

31 records · Page 2Linked to original sources

PubMed2004

Chondrocyte mechanotransduction: effects of compression on deformation of intracellular organelles and relevance to cellular biosynthesis.

OBJECTIVE: The effects of mechanical deformation of intact cartilage tissue on chondrocyte biosynthesis in situ have been well documented, but the mechanotransduction pathways that regulate such phenomena have not been elucidated completely. The goal of this study was to examine the effects of tissue deformation on the morphology of a range of intracellular organelles which play a major role in cell biosynthesis and metabolism. DESIGN: Using chemical fixation, high pressure freezing, and electron microscopy, we imaged chondrocytes within mechanically compressed cartilage explants at high magnification and quantitatively and qualitatively assessed changes in organelle volume and shape caused by graded levels of loading. RESULTS: Compression of the tissue caused a concomitant reduction in the volume of the extracellular matrix (ECM), chondrocyte, nucleus, rough endoplasmic reticulum, and mitochondria. Interestingly, however, the Golgi apparatus was able to resist loss of intraorganelle water and retain a portion of its volume relative to the remainder of the cell. These combined results suggest that a balance between intracellular mechanical and osmotic gradients govern the changes in shape and volume of the organelles as the tissue is compressed. CONCLUSIONS: Our results lead to the interpretive hypothesis that organelle volume changes appear to be driven mainly by osmotic interactions while shape changes are mediated by structural factors, such as cytoskeletal interactions that may be linked to extracellular matrix deformations. The observed volume and shape changes of the chondrocyte organelles and the differential behavior between organelles during tissue compression provide evidence for an important mechanotransduction pathway linking translational and post-translational events (e.g., elongation and sulfation of glycosaminoglycans (GAGs) in the Golgi) to cell deformation.

Animals↗

PubMed2004

Mechanical overload induces VEGF in cartilage discs via hypoxia-inducible factor.

VEGF (vascular endothelial growth factor) is not only one of the most important angiogenesis factors, but is involved also in inflammatory processes. Recent studies have shown that VEGF as well as its receptor VEGFR-2 are expressed on osteoarthritic chondrocytes, but not on normal adult chondrocytes. Since mechanical overload is one of the causative factors for osteoarthritis, we studied its effect on VEGF expression on bovine cartilage disks that were compressed once with a strain of 50% and a strain rate of 1/second. Under these conditions, control disks (without pressure) were completely negative for VEGF expression as evidenced by immunocytochemical stainings as well as by enzyme-linked immunosorbent assay (ELISA) measurements. In contrast, 4 days after mechanical overload, the cartilage disks were positive in both detection methods. In addition, after mechanical overload chondrocytes were strongly immunopositive for hypoxia-inducible factor-1alpha (HIF-1alpha), the limiting protein of the dimeric transcription factor HIF-1 that is known to induce VEGF expression. Furthermore, the matrix metalloproteases MMP-1, MMP-3, and MMP-13, could be easily detected in pressure-treated disks by immunohistochemistry whereas staining in controls was low or undetectable. The tissue inhibitors of metalloproteinases (TIMP-1 and -2) could be detected in controls but not in samples treated with mechanical overload. To prove that increased MMP or decreased TIMP expression could be a result of the autocrine action of VEGF on chondrocytes, we repeated the experiments in the presence of a specific inhibitor for the kinase activity of the VEGFR-2. This inhibitor was effective to reduce mechanically induced MMP-1, -3, and -13 immunostaining and to restore TIMP expression. Taking together, these findings indicate that VEGF is induced in chondrocytes by mechanical overload and mediates destructive processes in osteoarthritis as an autocrine factor.

Animals↗

PubMed2004

Three-dimensional head model simulation of transcranial magnetic stimulation.

This paper presents a finite element method used to evaluate the induced current density in a realistic model of the human head exposed to a time varying magnetic field. The tissue electric properties were varied to ascertain their influence on the induced currents. Current density magnitude and vector plots were generated throughout the tissue layers to determine the effects of tissue boundaries on the field. The current density magnitude correlated to the conductivity of the tissue in all the cases tested except where the tissue permittivity was raised to a level to allow for displacement currents. In this case, the permittivity of the tissue was the dominant factor. Current density components normal to the tissue interface were shown to exist in all solutions within the cortex contrary to the predictions of present models that rely on symmetrical geometries. Additionally, modifications in the cortical geometry were shown to perturb the field so that the site of activation could be altered in diseased patient populations. Finally, by varying the tissue permittivity values and the source frequency, we tested the effects of alpha dispersion theories on transcranial magnetic stimulation.

Adult↗

PubMed2004

In vitro gene transfer to chondrocytes and synovial fibroblasts by adenoviral vectors.

The major requirement of a successful gene transfer is the efficient delivery of an exogenous therapeutic gene to the appropriate cell type with subsequent high or regulated levels of expression. In this context, viral systems are more efficient than nonviral systems, giving higher levels of gene expression for longer periods. For the application of osteoarthritis (OA), gene products triggering anti-inflammatory or chondroprotective effects are of obvious therapeutic utility. Thus, their cognate genes are candidates for use in the gene therapy of OA. In this chapter, we describe the preparation, the use, and the effect of the transduction of chondrocytes or synovial fibroblasts with an adenoviral vector encoding the cDNA for glutamine: fructose-6-phosphate amidotransferase (GFAT). This is intended to serve as an example of a technology that can be used to evaluate the biological effects of overexpression of other cDNAs.

Adenoviridae↗

PubMed2003

Mechanical regulation of mitogen-activated protein kinase signaling in articular cartilage.

Articular chondrocytes respond to mechanical forces by alterations in gene expression, proliferative status, and metabolic functions. Little is known concerning the cell signaling systems that receive, transduce, and convey mechanical information to the chondrocyte interior. Here, we show that ex vivo cartilage compression stimulates the phosphorylation of ERK1/2, p38 MAPK, and SAPK/ERK kinase-1 (SEK1) of the JNK pathway. Mechanical compression induced a phased phosphorylation of ERK consisting of a rapid induction of ERK1/2 phosphorylation at 10 min, a rapid decay, and a sustained level of ERK2 phosphorylation that persisted for at least 24 h. Mechanical compression also induced the phosphorylation of p38 MAPK in strictly a transient fashion, with maximal phosphorylation occurring at 10 min. Mechanical compression stimulated SEK1 phosphorylation, with a maximum at the relatively delayed time point of 1 h and with a higher amplitude than ERK1/2 and p38 MAPK phosphorylation. These data demonstrate that mechanical compression alone activates MAPK signaling in intact cartilage. In addition, these data demonstrate distinct temporal patterns of MAPK signaling in response to mechanical loading and to the anabolic insulin-like growth factor-I. Finally, the data indicate that compression coactivates distinct signaling pathways that may help define the nature of mechanotransduction in cartilage.

Animals↗

PubMed2003

A comparative study of the inhibitory effects of interleukin-1 receptor antagonist following administration as a recombinant protein or by gene transfer.

Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret trade mark, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E2 production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1beta. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombinant human IL-1beta either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1beta produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombinant protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombinant protein became progressively less effective. This was particularly evident under conditions of continuous IL-1beta synthesis.

Animals↗

PubMed2003

Transport and binding of insulin-like growth factor I through articular cartilage.

This study focused on the role of insulin-like growth factor (IGF) binding proteins (IGFBPs) in cartilage on the transport and binding of IGF-I within the tissue. We have developed experimental and theoretical modeling techniques to quantify and contrast the roles of diffusion, binding, fluid convection, and electrical migration on the transport of IGF-I within cartilage tissue. Bovine articular cartilage disks were equilibrated in buffer containing 125I-IGF-I and graded levels of unlabeled IGF-I. Equilibrium binding, as measured by the uptake ratio of 125I-IGF-I in the tissue (free plus bound) to the concentration of labeled species in the buffer, was found to be consistent with a first-order reversible binding model involving one dominant family of binding sites within the matrix. Western ligand blots revealed a major IGF binding doublet around 23 kDa, which has been previously shown to coincide with IGFBP-6. Diffusive transport of 125I-IGF-I through cartilage was measured and found to be consistent with a diffusion-limited reaction theoretical model incorporating first-order reversible binding. Addition of excess amounts of unlabeled IGF-I during steady state transport of 125I-IGF-I resulted in release of bound 125I-IGF-I from the tissue, as predicted by the diffusion-reaction model. In contrast, addition of the low-affinity Des(1-3)IGF-I analog did not result in release of bound 125I-IGF-I. Application of electric current was used to augment transport of IGF-I through cartilage via electroosmosis and electrophoresis. Taken together, our results suggest that a single dominant substrate family, the high-affinity IGFBPs, is responsible for much of the observed binding of IGF-I within cartilage. The data suggest that intratissue fluid flow, such as that induced by mechanical loading of cartilage in vivo may be expected to enhance IGF transport by an order of magnitude and that this increment may help to counterbalance the restrictions encountered by the immobilization of IGFs by the binding proteins.

Animals↗

PubMed2003

Combined effects of dynamic tissue shear deformation and insulin-like growth factor I on chondrocyte biosynthesis in cartilage explants.

Biophysical forces and biochemical factors play crucial roles in the maintenance of the integrity of articular cartilage. In this study, we explored the effect of dynamic tissue shear deformation and insulin-like growth factor I (IGF-I) on matrix synthesis by chondrocytes within native cartilage explants. Dynamic tissue shear in the range of 0.5-6% strain amplitude at 0.1 Hz was applied to cartilage explants cultured in serum-free medium. Dynamic tissue shear above 1.5% strain amplitude significantly stimulated protein and proteoglycan synthesis, by maximum values of 35 and 25%, respectively, over statically held control specimens. In the absence of tissue shear, IGF-I augmented protein and proteoglycan synthesis up to twofold at IGF-I concentrations in the range of 100-300 ng/ml. When tissue shear and IGF-I stimuli were combined, matrix biosynthesis levels were significantly higher than the maximal effect caused by either stimulus alone. However, there was no significant interaction between tissue shear and IGF-I as determined by two-way ANOVA. We then quantified the effect of dynamic tissue shear on the transport of IGF-I into and within cartilage explants. [125I]IGF-I was added to the medium, and the levels of intratissue [125I]IGF-I were directly measured as a function of time over 48 h in the presence and absence of continuous dynamic shear strain. Dynamic shear did not alter the rate of uptake of [125I]IGF-I into the explants, suggesting that convective diffusion of [125I]IGF-I is negligible under the shear strain conditions used. This is in marked contrast to the enhancement of transport reported in response to uniaxial dynamic compression. Taken together, these data suggest that (1) the stimulatory effect of tissue shear is via mechanotransduction pathways and not by facilitated transport of biochemical factors and (2) chondrocytes may possess complementary signal transduction pathways for biophysical and biochemical factors leading to changes in metabolic activity.

Analysis of Variance↗

PubMed2003

Proteoglycan degradation after injurious compression of bovine and human articular cartilage in vitro: interaction with exogenous cytokines.

OBJECTIVE: Traumatic joint injury leads to an increased risk of osteoarthritis (OA), but the progression to OA is not well understood. We undertook this study to measure aspects of proteoglycan (PG) degradation after in vitro injurious mechanical compression, including up-regulation of enzymatic degradative expression and cytokine-stimulated degradation. METHODS: Articular cartilage tissue explants were obtained from newborn bovine femoropatellar groove and from adult normal human donor knee and ankle tissue. Following injurious compression of the cartilage, matrix metalloproteinase 3 (MMP-3) and MMP-13 messenger RNA (mRNA) expression levels were measured by Northern analysis, and PG loss to the medium after cartilage injury was measured in the presence and absence of added exogenous cytokine (interleukin-1alpha [IL-1alpha] or tumor necrosis factor alpha [TNFalpha]). RESULTS: During the first 24 hours after injury in bovine cartilage, MMP-3 mRNA levels increased 10-fold over the levels in control cartilage (n = 3 experiments), whereas MMP-13 mRNA levels were unchanged. PG loss was significantly increased after injury, but only by 2% of the total PG content and only for the first 3 days following injury. However, compared with injury alone or cytokine treatment alone, treatment of injured tissue with either 1 ng/ml IL-1alpha or 100 ng/ml TNFalpha caused marked increases in PG loss (35% and 54%, respectively, of the total cartilage PG content). These interactions between cytokine treatment and injury were statistically significant. In human knee cartilage, the interaction was also significant for both IL-1alpha and TNFalpha, although the magnitude of increase in PG loss was lower than that in bovine cartilage. In contrast, in human ankle cartilage, there was no significant interaction between injury and IL-1alpha. CONCLUSION: The cytokines IL-1alpha and TNFalpha can cause a synergistic loss of PG from mechanically injured bovine and human cartilage. By attempting to incorporate interactions with other joint tissues that may be sources of cytokines, in vitro models of mechanical cartilage injury may explain aspects of the interactions between mechanical forces and degradative pathways which lead to OA progression.

Adult↗

PubMed2003

Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy.

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.

Aggrecans↗

PubMed2003

Tissue-engineered versus native cartilage: linkage between cellular mechano-transduction and biomechanical properties.

Recent studies demonstrate that chondrocytes in native articular cartilage and in tissue-engineered constructs respond to mechanical stimuli through multiple regulatory pathways. Responses of the cells are manifested by intra- and intercellular signalling, alterations in transcription level, protein translation, post-translational modifications, and synthesis of intracellular and extracellular macromolecules. In addition, mechanical stimuli can alter the balance between anabolic and catabolic processes that are critically important to cell-mediated extracellular assembly and degradation of the tissue matrix and, therefore, to the survival of tissue engineered constructs. Chondrocyte mechanotransduction is therefore a critically important link to the biomechanical properties of native cartilage and to developing constructs. Since implanted cartilage repair tissue will be subjected to mechanical loads throughout its lifetime, it is essential that the resident cells respond appropriately to the range of static and dynamic compressive and shear deformations in vivo in a manner that enables adaptive remodelling and minimizes catabolic degradation. The in vivo environment should thereby signal tissue-specific maturation and integration processes in order to achieve the most appropriate tissue morphology and biomechanical function.

Animals↗

PubMed2002

Preservation and analysis of nonequilibrium solute concentration distributions within mechanically compressed cartilage explants.

Solute transport within articular cartilage is of central importance to tissue physiology, and may mediate effects of mechanical compression on cell metabolism. We therefore developed and applied a freeze-substitution method for fixation of cartilage explant disks which had been compressed axially during radial solute desorption. Dextrans were used as model solutes. Explant morphology was well preserved and nonequilibrium solute concentration distributions were stable for several hours at room temperature. For desorption from explants compressed statically to 0-46% strain, analysis of laser confocal images and comparison to a theoretical model permitted measurement of effective diffusivities. Results were consistent with previous studies suggesting a role for transport limitations in mediating the decreases of chondrocyte metabolic rates associated with static compression. In explants compressed dynamically (23+/-5% strain at 0.001 Hz), evidence was obtained for the augmentation of effective transport rate of 3 kDa dextrans by oscillatory interstitial fluid flows. This suggests that augmented solute transport may play a role in mediating the increases of chondrocyte metabolic rates associated with dynamic compression. Methods appear suitable for quantitative studies of transport within mechanically compressed cartilage-like tissues, and may be valuable for identification of loading environments which optimize solute transport in tissue engineering applications.

Animals↗

PubMedDate not supplied

Evaluation of medium supplemented with insulin-transferrin-selenium for culture of primary bovine calf chondrocytes in three-dimensional hydrogel scaffolds.

Insulin-transferrin-selenium (ITS) was investigated as a complete or partial replacement for fetal bovine serum (FBS) during in vitro culture of bovine calf chondrocytes in hydrogel scaffolds. Chondrocyte-seeded agarose and self-assembling peptide hydrogels were maintained in Dulbecco's modified Eagle's medium plus 10% FBS, 1% ITS plus 0.2% FBS, or 1% ITS and evaluated for biosynthesis, cell division, and surface outgrowth of fibroblastic-like cells and fibrous capsule formation over several weeks of culture. In peptide hydrogels, cells cultured in ITS plus 0.2% FBS medium exhibited high rates of biosynthesis and showed similar cell division trends as seen in 10% FBS cultures. ITS medium alone did not support glycosaminoglycan accumulation beyond 5 days of culture, and cell division was less than that in both serum-containing cultures. Extensive cellular outgrowth and fibrous capsule formation were observed in 10% FBS medium, whereas little outgrowth was observed in ITS plus 0.2% FBS and none was seen in ITS medium alone. In agarose hydrogels, chondrocyte biosynthesis and cell division in ITS medium were similar to that in 10% serum culture over 5 weeks, and cellular outgrowth was eliminated. Taken together, ITS was suitable as a partial (peptide) or complete (agarose) substitute for serum, and also provided the benefit of reducing or eliminating cell outgrowth and fibrous capsule formation on the hydrogel surface.

Animals↗