Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins.

In this study, we investigated the biosynthetic response of full thickness, adult bovine articular cartilage explants to 45 h of static and cyclic unconfined compression. The cyclic compression of articular cartilage resulted in a progressive consolidation of the cartilage matrix. The oscillatory loading increased protein synthesis ([35S]methionine incorporation) by as much as 50% above free swelling control values, but had an inhibitory influence on proteoglycan synthesis ([35SO4] incorporation). As expected, static compression was associated with a dose-dependent decrease in biosynthetic activity. ECM oligomeric proteins which were most affected by mechanical loading were fibronectin and cartilage oligomeric matrix protein (COMP). Static compression at all amplitudes caused a significant increase in fibronectin synthesis over free swelling control levels. Cyclic compression of articular cartilage at 0.1 Hz and higher was consistently associated with a dramatic increase in the synthesis of COMP as well as fibronectin. The biosynthetic activity of chondrocytes appears to be sensitive to both the frequency and amplitude of the applied load. The results of this study support the hypothesis that cartilage tissue can remodel its extracellular matrix in response to alterations in functional demand.     

Dynamic compression of rabbit adipose-derived stem cells transfected with insulin-like growth factor 1 in chitosan/gelatin scaffolds induces chondrogenesis and matrix biosynthesis

Articular cartilage is routinely subjected to mechanical forces and growth factors. Adipose-derived stem cells (ASCs) are multi-potent adult stem cells and capable of chondrogenesis. In the present study, we investigated the comparative and interactive effects of dynamic compression and insulin-like growth factor-I (IGF-I) on the chondrogenesis of rabbit ASCs in chitosan/gelatin scaffolds. Rabbit ASCs with or without a plasmid overexpressing of human IGF-1 were cultured in chitosan/gelatin scaffolds for 2 days, then subjected to cyclic compression with 5% strain and 1 Hz for 4 h per day for seven consecutive days. Dynamic compression induced chondrogenesis of rabbit ASCs by activating calcium signaling pathways and up-regulating the expression of Sox-9. Dynamic compression plus IGF-1 overexpression up-regulated expression of chondrocyte-specific extracellular matrix genes including type II collagen, Sox-9, and aggrecan with no effect on type X collagen expression. Furthermore, dynamic compression and IGF-1 expression promoted cellular proliferation and the deposition of proteoglycan and collagen. Intracellular calcium ion concentration and peak currents of Ca(2+) ion channels were consistent with chondrocytes. The tissue-engineered cartilage from this process had excellent mechanical properties. When applied together, the effects achieved by the two stimuli (dynamic compression and IGF-1) were greater than those achieved by either stimulus alone. Our results suggest that dynamic compression combined with IGF-1 overexpression might benefit articular cartilage tissue engineering in cartilage regeneration.     

A role for the interleukin-1 receptor in the pathway linking static mechanical compression to decreased proteoglycan synthesis in surface articular cartilage

Loading of articular cartilage during weight bearing is essential for the maintenance of cartilage function. Although certain cyclic loading protocols stimulate extracellular matrix synthesis, constant or static compression decreases proteoglycan and collagen synthesis in cartilage explants. The goal of this study was to determine whether the compression-induced decrease in proteoglycan synthesis involves an interleukin-1 (IL-1) signaling pathway. Cartilage explants were compressed 50% in the presence of IL-1 receptor antagonist (IL-1ra), and the incorporation of [35S]sulfate into macromolecules was measured. IL-1ra increased sulfate incorporation in compressed cartilage but not in cartilage maintained at the in situ thickness (0% compression). IL-1alpha and IL-1beta mRNAs were detected in cartilage compressed 50% for at least 3h, while nitric oxide synthase II mRNA was only detected in cartilage compressed 50% for 6h. The data support a role for the IL-1 receptor in the pathway linking static compression to reduced proteoglycan synthesis.     

Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro

This study investigates the separate and combined effects of IGF-1 and mechanical loads on chondrocytes in elderly human femoral head articular cartilage. Full depth biopsies of articular cartilage were subjected to either no load, static or cyclic (2 s on/2 s off) loading in unconfined compression at a stress of 1 MPa for 48 h with or without IGF-1 (300 ng ml(-1)). Chondrocyte biosynthetic activity was measured using 35S-sulphate and 3H-leucine during the last 24 h of loading. IGF-1 alone increased the rates of isotope incorporation, by 80% for 35S-SO4 and 40% for 3H-leucine, whereas loading alone reduced matrix biosynthesis. Applying load (cyclic or static) in the presence of IGF-1 returned the incorporation rates to their unstimulated levels. This study suggests elderly human articular cartilage is responsive to stimulation by IGF-1 but mechanical factors seem to act sufficiently strongly in the opposite direction to cancel this response.     

Extracellular Calcium-Induced Neuroblastoma Cell Differentiation: Involvement of Phosphatidylinositol Turnover

The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10-25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium-dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second-messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6-10 h after addition of calcium), and increase in intracellular calcium levels (12-24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10-25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic-like junctions; this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.     

Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants

The effects of mechanical compression of calf cartilage explants on the catabolism and loss into the medium of proteoglycans and proteins radiolabeled with [35S]sulfate and [3H]proline were examined. A single 2- or 12-h compression of 3-mm diameter cartilage disks from a thickness of 1.25 to 0.50 mm, or slow cyclic compression (2 h on/2 h off) from 1.25 mm to 1.00, 0.75, or 0.50 mm for 24 h led to transient alterations and/or sustained increases in loss of radiolabeled macromolecules. The effects of imposing or removing loads were consistent with several compression-induced physical mediators including fluid flow, diffusion, and matrix disruption. Cyclic compression induced convective fluid flow and enhanced the loss of 35S- and 3H-labeled macromolecules from tissue into medium. In contrast, prolonged static compression induced matrix consolidation and appeared to hinder the diffusional transport and loss of 35S- and 3H-labeled macromolecules. Since high amplitude cyclic compression led to a sustained increase in the rate of loss of 3H- and 35S-labeled macromolecules that was accompanied by an increase in the rate of loss of [3H]hydroxyproline residues and an increase in tissue hydration, such compression may have caused disruption of the collagen meshwork. The 35S-labeled proteoglycans lost during such cyclic compression were of smaller average size than those from controls, but contained a similarly low proportion (approximately 15%) that could form aggregates with excess hyaluronate and link protein. The size distribution and aggregability of the remaining tissue proteoglycans and 35S-labeled proteoglycans were not markedly affected. The loss of tissue proteoglycan paralleled the loss of 35S-labeled macromolecules. This study provides a framework for elucidating the biophysical mechanisms involved in the redistribution, catabolism, and loss of macromolecules during cartilage compression.     

Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage

The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0-50% static compression, and the transient and steady-state incorporation of [(3)H]proline and [(35)S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0-300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [(3)H]proline and [(35)S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of (125)I-labeled IGF-I into cartilage explants. Static compression decreased both [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (tau) for IGF-I stimulation of cartilage explants was approximately 24 h, while tau for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter tau for (125)I-labeled IGF-I transport into cartilage but decreased the concentration of (125)I-labeled IGF-I in the tissue at equilibrium.     

Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation.

Overcoming the limited ability of articular cartilage to self-repair may be possible through tissue engineering. However, bioengineered cartilage formed using current methods does not match the physical properties of native cartilage. In previous studies we demonstrated that mechanical stimulation improved cartilage tissue formation. This study examines the mechanisms by which this occurs. Application of uniaxial, cyclic compression (1 kPa, 1 Hz, 30 min) significantly increased matrix metalloprotease (MMP)-3 and MMP-13 gene expression at 2 h compared to unstimulated cells. These returned to constitutive levels by 6 h. Increased MMP-13 protein levels, both pro- and active forms, were detected at 6 h and these decreased by 24 h. This was associated with tissue degradation as more proteoglycans and collagen had been released into the culture media at 6 h when compared to the unstimulated cells. This catabolic change was followed by a significant increase in type II collagen and aggrecan gene expression at 12 h post-stimulation and increased synthesis and accumulation of these matrix molecules at 24 h. Mechanical stimulation activated the MAP kinase pathway as there was increased phosphorylation of ERK1/2 and JNK as well as increased AP-1 binding. Mechanical stimulation in the presence of the JNK inhibitor, SP600125, blocked AP-1 binding preventing the increased gene expression of MMP-3 and -13 at 2 h and type II collagen and aggrecan at 12 h as well as the increased matrix synthesis and accumulation. Given the sequence of changes, cyclic compressive loading appears to initiate a remodelling effect involving MAPK and AP-1 signalling resulting in improved in vitro formation of cartilage.     

Activation of chondrocytes calcium signalling by dynamic compression is independent of number of cycles

Mechanical loading is necessary for the development and maintenance of healthy articular cartilage through the control of extracellular matrix synthesis and catabolism. However, the underlying process of chondrocyte mechanotransduction remains unclear. This study examined the influence of cyclic compression on intracellular calcium (Ca(2+)) signalling within isolated articular chondrocytes cultured in agarose constructs. A validated experimental system was developed for applying controlled cyclic cell deformation. Cell-agarose constructs were subjected to 1Hz cyclic compression between 0 and 10% gross strain for 1, 10, 100 or 300 cycles. The cells were subsequently visualised for 300s in the unstrained state using confocal microscopy and the Ca(2+) indicator, Fluo-4 AM. Within unloaded control constructs, a sub-population of approximately 50% of chondrocytes exhibited characteristic spontaneous Ca(2+) transients each lasting approximately 40-60s. Cyclic compression, for only 1 cycle, significantly up-regulated the percentage of cells exhibiting Ca(2+) transients in the subsequent 5min period (p<0.05). Increasing the number of cycles to 10 or 100 had no additional effect. The up-regulated Ca(2+) signalling was maintained for up to 5min before returning to basal levels. By contrast, 300 cycles were followed by Ca(2+) signalling that was not significantly different from that in unloaded controls. However, this response was shown to be due to the increased time following the start of compression. In conclusion, this study indicates that chondrocyte Ca(2+) signalling is stimulated by dynamic compression, probably mediated by cyclic cell deformation. The overall response appears to be independent of the number of cycles or duration of cyclic compression. The sustained up-regulation of Ca(2+) signalling after 1, 10 or 100 cycles suggests the involvement of an autocrine-paracrine signalling mechanism. Furthermore, the reduced response following 300 cycles indicates a possible receptor desensitisation mechanism. Therefore, Ca(2+) signalling may be part of a mechanotransduction pathway through which chondrocyte populations can modulate their metabolic activity in response to changing mechanical stimuli.     

Cyclic stretch decreases TRPC4 protein and capacitative calcium entry in rat vascular smooth muscle cells

We investigated whether cyclic stretch affects TRPC4 or TRPC6 expression and calcium mobilization in cultured vascular smooth muscle cells. In aortic and mesenteric smooth muscle cells isolated from male Sprague-Dawley rats, TRPC4 expression was decreased after 5 h stretch and remained suppressed through 24 h stretch. After removal of the stretch stimulus, TRPC4 expression recovered within 2 h. Stretch did not affect TRPC6 expression. Stretch also decreased capacitative calcium entry, while agonist-induced calcium influx was increased. Similar results were obtained in primary aortic smooth muscle cells. TRPC4 mRNA levels were not decreased in response to mechanical strain. TRPC4 downregulation was also achieved by increasing extracellular calcium and was attenuated by gadolinium and MG132, suggesting that TRPC4 protein is regulated by intracellular calcium concentration and/or the ubiquitin-proteasome pathway. These data suggest that stretch-induced downregulation of TRPC4 protein expression and capacitative calcium entry may be a protective mechanism to offset stretch-induced increases in intracellular calcium.     

Stimulation of inorganic pyrophosphate elaboration by cultured cartilage and chondrocytes

Inorganic pyrophosphate elaboration by articular cartilage may favor calcium pyrophosphate dihydrate crystal deposition. Frequently crystal deposits form in persons affected with metabolic diseases. The cartilage organ culture system was used to model these metabolic conditions while measuring the influence on extracellular pyrophosphate elaboration. Alterations of ambient pH, thyroid stimulating hormone levels, and parathyroid hormone levels did not change pyrophosphate accumulation in the media. However, subphysiologic ambient calcium concentrations (25, 100, 500 microM) increased pyrophosphate accumulation about chondrocytes 3- to 10-fold. Low calcium also induced release of [14C]adenine-labeled nucleotides from chondrocytes, potential substrates for generation of extracellular pyrophosphate by ectoenzymes. Exposing cartilage to 10% fetal bovine serum also enhanced by 50% the egress of inorganic pyrophosphate from the tissue.     

Static compression is associated with decreased diffusivity of dextrans in cartilage explants

The chondrocytes of adult articular cartilage rely upon transport phenomena within their avascular extracellular matrix for many biological activities. Therefore, changes in matrix structure which influence cytokine transport parameters may be an important mechanism involved in the chondrocyte response to tissue compression. With this hypothesis in mind, partitioning and diffusion of 3-, 10-, and 40-kDa dextrans conjugated to tetramethylrhodamine, and 430-Da tetramethylrhodamine itself, were measured within statically compressed bovine articular cartilage explants using a novel experimental apparatus and desorption fluorescence method. Partitioning and diffusion were examined as functions of solute molecular weight and matrix proteoglycan density, and diffusion was measured versus static compression up to 35% volumetric strain. In general, partition coefficients and diffusivities were found to decrease with increasing solute molecular weight. In addition, for a given solute, diffusivities decreased significantly with increasing static compression. Results therefore suggest a possible role for transport limitations of relatively large molecular weight solutes within the extracellular matrix in mediating the biological response of chondrocytes to cartilage compression.     

G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.     

Static and dynamic compression regulate cartilage metabolism of PRoteoGlycan 4 (PRG4)

The boundary lubrication function of articular cartilage is mediated in part by molecules at the articular surface and in synovial fluid, encoded by Prg4. The objective of this study was to determine whether static and dynamic compression regulate PRG4 biosynthesis by cartilage explants. Articular cartilage disks were harvested to include the articular surface from immature bovines. Some disks were subjected to 24 h (day 1) of loading, followed by 72 h (days 2-4) of free-swelling culture to assess chondrocyte responses following unloading. Loading consisted of 6 or 100 kPa of static compression, with or without superimposed dynamic compression (10 or 300 kPa peak amplitude, 0.01 Hz). Other disks were cultured free-swelling as controls. PRG4 secretion into culture medium was inhibited by all compression protocols during day 1. Following unloading, cartilage previously subjected to dynamic compression to 300 kPa exhibited a rebound effect, secreting more PRG4 than did controls, while cartilage previously subjected to 100 kPa static loading secreted less PRG4. Immunohistochemistry revealed that all compression protocols also affected the number of cells expressing PRG4. The paradigm that mechanical stimuli regulate biosynthesis in cartilage appears operative not only for load bearing matrix constituents, but also for PRG4 molecules mediating lubrication.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.