Influence of strain on proteoglycan synthesis by valvular interstitial cells in three-dimensional culture

Differently loaded regions of the mitral valve contain distinct amounts and types of proteoglycans (PGs); these PG profiles are altered in abnormal loading and disease conditions. We developed an in vitro three-dimensional model to analyze PGs secreted by valvular interstitial cells (VICs) isolated from distinct regions of porcine mitral valves (leaflet or chordae) and subjected to either biaxial or uniaxial mechanical constraints. In addition, the PGs, DNA and collagen content of the collagen gels was monitored over time. All three PGs previously found in heart valves (decorin, biglycan and versican) were present in the collagen gels and the conditioned medium. Compared to unconstrained gels, the constrained collagen gels (whether biaxially or uniaxially loaded) retained more decorin and biglycan but less versican. However, the conditioned medium from constrained collagen gels contained higher amounts of all three PGs than did medium from unconstrained gels. Constrained collagen gels containing leaflet cells retained more decorin and biglycan than did those containing chordal cells. DNA content was maintained early in the culture period but was reduced by 55-80% after 7 days, whereas PG synthesis increased over time. At the end of the culture period, the cell density was highest in the biaxial region of gels seeded with leaflet cells. In contrast, collagen content in both constrained and unconstrained gels remained consistent over culture duration. This study provides valuable information about the role of applied loading on proteoglycan segregation, which should aid in tissue engineering applications and for understanding valve biology and pathology.     

Valve proteoglycan content and glycosaminoglycan fine structure are unique to microstructure, mechanical load and age: Relevance to an age-specific tissue-engineered heart valve

This study characterized valve proteoglycan and glycosaminoglycan composition during development and aging. This knowledge is important for the development of age-specific tissue-engineered heart valves as well as treatments for age-specific valvulopathies. Aortic valves and mitral valves from first-third trimester, 6-week, 6-month and 6-year-old pigs were examined using immunohistochemistry for versican, biglycan, decorin and hyaluronan, as well as elastin and fibrillin. The fine structure of glycosaminoglycans was examined by fluorophore-assisted carbohydrate electrophoresis. Decorin expression was strongest in the 6-year-old valves, particularly in the aortic valve spongiosa. The quantity of iduronate was also highest in the 6-year-old valves. The central tensile-loading region of the anterior mitral leaflet demonstrated reduced glycosaminoglycan content, chain length and hydration and a larger fraction of 4-sulfated iduronate and lower fraction of 6-sulfation. With age, the anterior leaflet center showed a further increase in 4-sulfated iduronate and decrease in 6-sulfation. In contrast, the anterior leaflet free edge showed decreased iduronate and 4-sulfated glucuronate content with age. The young aortic valve was similar to the mitral valve free edge with a higher concentration of glycosaminoglycans and 6-rather than 4-sulfation, but aged to resemble the mitral anterior leaflet center, with an increase in 4-sulfated iduronate content and a decrease in the 6-sulfation fraction. Elastin and fibrillin often co-localized with the proteoglycans studied, but elastin co-localized most specifically with versican. In conclusion, composition and fine structure changes in valve proteoglycans and glycosaminoglycans with age are complex and distinct within valve type, histological layers and regions of different mechanical loading.     

Time-dependent mechanical properties of aortic valve cusps: Effect of glycosaminoglycan depletion

Aortic valve (AV) performance is closely linked to its structural components. Glycosaminoglycans (GAGs) are thought to influence the time-dependent properties of living tissues. This study investigates the effect of GAGs on the viscoelastic behaviour of the AV. Fresh porcine AV cusps were either treated enzymatically to remove GAGs or left untreated (control). The specimens were tested for stress relaxation and tensile properties under equibiaxial load conditions. The stress relaxation curves were fitted using a double exponential decay equation and the early relaxation constant (τ₁) and late relaxation constant (τ₂) calculated for each specimen. Immunohistochemistry confirmed the successful depletion of both sulphated and non-sulphated GAGs from the AV cusps. A statistical increase in τ₁ was found for both the radial and circumferential directions between the control and –GAGs group (radial, control 17.37 s vs. –GAGs 25.65 s; circumferential, control 21.47 s vs. –GAGs 27.37 s). It was also found that τ₁ differed between the two directions for the control group but not after GAG depletion (control, radial 17.37 s vs. circumferential 21.47 s; –GAGs, radial 25.65 s vs. circumferential 27.37 s). No effect on stiffness was found. The results showed that the presence of GAGs influences the mechanical viscoelastic properties of the AV but has no effect on the stiffness. The natural anisotropy, which reflects the relaxation kinematics, is lost after GAG depletion.     

A stimulation unit for the application of mechanical strain on tissue engineered anulus fibrosus cells: a new system to induce extracellular matrix synthesis by anulus fibrosus cells dependent on cyclic mechanical strain

A bioreactor system consisting of a multifunctional stimulation unit and common 6-well culture plate is introduced to activate extracellular matrix synthesis in intervertebral disc cells due to cyclic mechanical strain. The developed stimulation unit is sterilizable and reusable. It is viable for cultivation and mechanical stimulation of cartilage tissue and tissue engineered cell matrix constructs in combination with the common 6-well culture plate. The custom made device allows long-term cultivations in batch- or continuous operation mode. Manual handling and thereby the risk of contamination is reduced. Sampling, changing the medium, and addition of supplements are easily performed from the connected conditioning vessel. This bioreactor system enables stimulation of different samples independently during one run. For the work presented here anulus fibrosus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs. Over a period of 14 days the constructs were subjected to 10% compression under cyclic mechanical pressure with a frequency of 0.1 Hz. Afterwards the constructs were biochemically examined for hydroxyproline and sulphated glycosaminoglycanes. These proven constituents of extracellular matrix were found to be released depending on the applied compressive strain.     

Enhancement of nitric oxide and proteoglycan synthesis due to cyclic tensile strain loaded on chondrocytes attached to fibronectin

Objective:Mechanical stress is an essential factor in the pathogenesis of osteoarthrosis. We sought to determine whether the strain-mediated alteration in proteoglycan (PG) synthesis was modulated by nitric oxide (NO) synthesis.Methods:Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [³⁵S] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used.Results:Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced.Conclusions:Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis.Abbreviations:Inducible NO synthase (iNOS), proteoglycan (PG), L-N-monomethylarginine (L-NMA), nitric oxide (NO)Received 7 July 2003; returned for revision 23 September 2003; accepted by W. B. van den Berg 23 December 2003     

Effect of cytokines and anti-arthritic drugs on glycosaminoglycan synthesis by bovine articular chondrocytes

The effect of cytokines (interleukin-1, interleukin-1, and tumor necrosis factor ) and several antiarthritic drugs on glycosaminoglycan synthesis and secretion into medium by bovine articular chondrocytes was examined. Sulfated glycosaminoglycans (S-GAG) were measured by a modified 1,9-dimethylmethylene blue (DMB) dye binding assay. Hyaluronate (HA) was measured by an inhibition ELISA based on specific binding to a proteoglycan. All three cytokines caused a dose-dependent decrease in S-GAG production and a dose-dependent increase in HA production. Non-steroidal antiinflammatory drugs (NSAIDs, indomethacin, naproxen, and piroxicam, 1M) could not reverse the effect of IL-1 on inhibiting S-GAG and stimulating HA synthesis. The anti-inflammatory steroid (dexamethasone, 1M) depressed HA synthesis by 50–70% in the absence or presence of IL-1. Dexamethasone depressed S-GAG synthesis by 20–30% in the absence or presence of IL-1. Therefore, none of the tested anti-rheumatic drugs reversed the cytokine mediated changes in glycosaminoglycan synthesis by bovine chondrocytes.     

The effect of cyclic mechanical strain on activation of dendritic cells cultured on adhesive substrates

Dendritic cells (DCs), key regulators of tolerance and immunity, have been found to reside in mechanically active tissues such as the interior layers of the arterial wall, which experience cyclic radial wall strain due to pulsatile blood flow. Although experimentally difficult to determine in vivo, it is reasonable to postulate DCs experience the mechanical forces in such mechanically active tissues. However, it is currently unknown how DCs respond to cyclic mechanical strain. In order to explore the hypothesis that DCs are responsive to mechanical strain, DCs were cultured in vitro on pre-adsorbed adhesive proteins (e.g., laminin, collagen, fibrinogen) and 1 Hz cyclic strain was applied for various durations and strain magnitudes. It was determined that a strain magnitude of 10% and 24 h duration adversely affected DC viability compared to no-strain controls, but culture on certain adhesive substrates provided modest protection of viability under this harsh strain regime. In contrast, application of 1 h of 1 Hz cyclic 3% strain did not affect DC viability and this strain regime was used for the remaining experiments for quantifying DC activation and T-cell priming capability. Application of 3% strain increased expression of stimulatory (MHC-II) and costimulatory molecules (CD86, CD40), and this effect was generally increased by culture on pre-coated adhesive substrates. Interestingly, the cytokine secretion profile of DCs was not significantly affected by strain. Lastly, strained DCs demonstrated increased stimulation of allogeneic T-cell proliferation, in a manner that was independent of the adhesive substrate. These observations indicate generation of a DC consistent with what has been described as a semi-mature phenotype. This work begins elucidating a potential role for DCs in tissue environments exposed to cyclic mechanical forces.     

Effect of cyclic strain on the mechanical behavior of virgin ultra-high molecular weight polyethylene

Ultra High Molecular Weight Polyethylene (UHMWPE) is a polymeric material employed in critical biomedical applications. Knowledge of its mechanical behavior is essential in order to obtain accurate prediction of stresses and deformations in real components, in particular when cyclic loading is considered. In the present research the effects of alternating and pulsating cyclic strain on the mechanical response of UHMWPE were studied by means of an experimental procedure based on tests carried out in strain control at different mean cyclic strain levels. During the tests the temperature increase due to hysteretic heating was controlled by means of a compressed air cooling apparatus specifically devised. By taking advantage of the possibility to control and stabilize temperature, cyclic steady-state mechanical response was investigated at room temperature and at 37 and 50 °C, comparing the effects of alternating and pulsating loading cycles. A transient thermal analysis using the finite element method (FEM) was also carried out to analyze temperature distribution within the specimen. UHMWPE exhibited cyclic softening as a result of a thermal contribution due to temperature increase and of a mechanical contribution related to the effects of applied load on the microstructure. The material exhibited different peak stress percent reductions for pulsating and alternating loading and during tensile and compressive loading phases. For pulsating tests significant cyclic mean stress relaxation was also observed. Based on the experimental procedure described the cyclic curve was determined as a function of temperature and fitted with a Ramberg–Osgood type constitutive equation, in which material parameters are temperature dependent. In this way the combined effects of temperature rises, such as those that might occur in biological environments or due to frictional heating, and mechanical loads could effectively be taken into account for constitutive modeling purposes of cyclic mechanical behavior of UHMWPE.     

Phonographic detection of mechanical heart valve thrombosis

The formation of thrombotic deposits affects the functionality of mechanical prosthetic heart valves; as a consequence, mechanical valves thrombosis needs early diagnosis to prevent thromboembolic events. This paper compares the acoustic signals produced by two commercial bileaflet mechanical heart valves in the closing phase to detect the presence of thrombi. The closing sounds were recorded in vitro by means of a phonocardiographic device under different hydrodynamic conditions. Thrombotic deposits of different weight and shape were applied onto the valve leaflet and the annular housing, until the movement of one leaflet was completely blocked. From the acoustic signals, the corresponding spectra were calculated and four diagnostic frequency bands were identified: their comparison allowed detecting malfunctioning valves because of the presence of thrombotic formations.     

Cavitation potential of mechanical heart valve prostheses

Just like technical check valves, the function of mechanical heart valve prostheses may presumably also lead to cavitation effects during valve closure. Due to the waterhammer effect, cavitation may primarily occur in the mitral position leading to high mechanical loading of the valve itself and of corpuscular blood elements. Ten different types of commercial mechanical heart valves were investigated in the mitral position of a pulsatile mock loop, to detect cavitation thresholds under physiologically similar conditions by cinematographic techniques. Almost all these valve prostheses show cavitation up to a ventricular pressure gradient of 5000 mmHg/s. The threshold depends on valve type and size and is sometimes within the physiological range below 2000 mmHg/s. Visible cavitation bubbles with a diameter of up to 1.8 mm and a collapse time of less than 0.1 ms suggest that vapour cavitation may play an important role for material and blood damage in mechanical heart valve prostheses.     

Hyaluronan reversed proteoglycan synthesis inhibited by mechanical stress: possible involvement of antioxidant effect

Introduction  

Abnormal mechanical stress loaded on the cartilage leads to the osteoarthritis (OA). Although intraarticular hyaluronan (HA) injection is an effective treatment for OA, the underlying mechanism has not been made clear.     

Repetitive mechanical strain suppresses macrophage uptake of immunoglobulin G complexes and enhances cyclic adenosine monophosphate synthesis.

Uptake of immunoglobulin G (IgG) complexes by macrophages (M phi) may play an important role in disease states characterized by increased levels of circulating immune complexes. In sites such as the glomerular mesangium M phi may be subjected to repetitive mechanical strain, although in vitro studies of M phi endocytosis are typically carried out with cells grown on rigid surfaces. We undertook the present study to determine whether repetitive mechanical strain could modulate M phi endocytosis of IgG complexes. IgG complex uptake was significantly diminished in M phi that were subjected to repetitive mechanical strain using parameters corresponding to peak and minimal intraglomerular pressures compared with control, and uptake varied according to the amount of mechanical strain applied. There was no significant difference in surface binding of IgG between M phi subjected to strain and those not. Mechanical strain did not significantly influence the rate of IgG complex degradation. Inhibition of nitric oxide synthase and guanylate cyclase activity did not alter the effect of mechanical strain, although this effect was potentiated by 3-isobutyl-1-methylxanthine (IBMX). Angiotensin II, which has been shown to reduce adenosine 3',5'-cyclic monophosphate (cAMP) production in M phi, significantly attenuated the suppressive effect of mechanical strain on IgG complex uptake as well as another inhibitor of cAMP generation, indomethacin. Enzyme immunoassay demonstrated significantly enhanced levels of cAMP in M phi that were subjected to mechanical strain compared with control, an effect that was potentiated by IBMX and attenuated by angiotensin II and indomethacin. These results demonstrate that repetitive mechanical strain significantly reduces IgG complex uptake by M phi, most likely by enhancing cAMP synthesis. Such an effect might play a significant role in macromolecule handling by M phi in sites in which they are subjected to repetitive mechanical deformation such as the glomerular mesangium.     

Effect of thyroid hormone deficiency on proteoglycan synthesis by human skin fibroblast cultures

Proteoglycans and hyaluronic acid synthesized by human skin fibroblasts in culture were characterized, and the effect of thyroid hormone deficiency was examined. The fibroblasts in culture synthesize hyaluronic acid, dermatan sulfate (DS) proteoglycans and heparan sulfate (HS) proteoglycans. Hyaluronic acid is almost exclusively secreted into the medium. Among the proteoglycans synthesized during 24 h label, about 70% were secreted into the medium and the remaining 30% were associated with the cell layer. About 70% of proteoglycans secreted into the medium contained DS and the remaining 30% contained HS. For cell-associated proteoglycans, 60% contained HS and the remainder contained DS. The size distributions of the glycosaminoglycans from both DS and HS proteoglycans were similar, with an average Mr of approximately 30,000. Incubation of fibroblasts in thyroid hormone deficient medium increased net synthesis of hyaluronic acid (approximately 50%) and all species of proteoglycans (approximately 85%). 3H/35S ratios in the chondroitin 4-sulfate disaccharide isolated with HPLC were not altered in thyroid hormone deficient cultures, indicating that the specific activity of 3H in UDP-N-acetylhexosamine precursors did not change. The increased incorporation of 3H into hyaluronic acid and of 3H and 35S into DS and HS proteoglycans thus indicates increased net synthesis. Degradation of cell-associated proteoglycans was not influenced by thyroid hormone deficiency.     

Cyclic mechanical strain decreases the DNA synthesis of vascular smooth muscle cells

In vivo, smooth muscle cells of the vascular wall are rhythmically stretched by the arterial pulse. Here, we test the hypothesis that rhythmical stretch is important for suppressing the growth of vascular smooth muscle (vsm) cells. DNA-synthesis rate, cell number, metabolic activity, and cell death were compared between rhythmically stretched and non-stretched vsm cells from the rat embryonic aortic A10 cell line. Rhythmical stretch (0.5 Hz, 5% elongation, 48 h) did not induce vsm cell proliferation, that is the vsm cell number was constant. Cell damage or necrosis was excluded because the release of lactate dehydrogenase (LDH) was identical. The low rate of apoptosis (0.2%) was not different between stretched cells and control cells. Stretch significantly reduced the DNA-synthesis rate [measured as incorporation of 5-bromo-2'-deoxyuridine (BrdU)] in a time-dependent manner. BrdU incorporation was decreased by 32% after 24 h of cyclic stretching and was further diminished to 50% after 48 h of strain. Metabolic activity (measured by Wst-1 cleavage) was only modestly influenced. The stretch-induced decrease in DNA synthesis was independent of the extracellular matrix. No differences were detected when laminin- or pronectin-coated membranes were used instead of collagen-coated membranes. The effect of stretch was unlikely to be mediated by secretion of an unknown "factor", because vsm cells incubated with medium conditioned by stretched cells did not show a significant decrease in BrdU uptake. The results support the idea that rhythmical stretch is important to keep the rate of DNA synthesis and thereby the proliferation of vsm cells at a low level.     

Human tumor cells in culture stimulate glycosaminoglycan synthesis by human skin fibroblasts

The human tumor cell lines, MM-96, FME, HCT-8, HT-29, MCF-7 and T-47D, in culture produced a factor or factors able to stimulate glycosaminoglycan (GAG) synthesis in human skin fibroblasts (HF). Conditioned growth media from the melanoma MM-96 and the colon carcinoma HT-29 produced a 10- and 8-fold stimulation of HF GAG synthesis, respectively, with an even larger stimulation of hyaluronic acid. Conditioned media from the melanoma FME and the breast carcinomas MCF-7 and T-47D stimulated GAG synthesis 2-fold, whereas media from the colon carcinoma HCT-8 gave a variable response often with no effect on GAG levels. Conditioned media from HF cultures had no effect on tumor cell GAG synthesis. Coculture of tumor cells and HF also resulted in increased GAG synthesis, and the degree of stimulation was similar to that with the conditioned media. Tumor cell-conditioned media were also effective in stimulating GAG synthesis by porcine smooth muscle cells and by chick embryo fibroblasts in culture, although the increase in GAG synthesis was much less than with HF cultures. These findings support the concept that the stromal desmoplasia characteristic of many growing and invasive tumors in vivo arises by tumor cell modulation of GAG synthesis by surrounding normal connective tissue cells.     

Effect of cyclic nucleotides on DNA synthesis in mouse lymphoid cells.

The effect of eight cyclic purine and cyclic pyrimidine nucleotides on DNA synthesis on mouse lymphoid cells was investigated. Two out of eight compounds tested, namely 2', 3'-cyclic guanosine monophosphate (2', 3'-cGMP) as well as 3', 5'-cyclic guanosine monophosphate (3', 5'-cGMP), stimulate thymidine incorporation in all types of lymphocytes tested. The stimulatory activity of the cyclic guanosine nucleotides as well as the effects of lectins could be antagonized by 3', 5'-cyclic adenosine monophosphate (3', 5'-cAMP). 2', 3'-cGMP seems to stimulate preferentially mature T-cells while 3', 5'-cGMP preferentially acts on B-cells.     

Mechanisms of bioprosthetic heart valve failure: fatigue causes collagen denaturation and glycosaminoglycan loss.

Bioprosthetic heart valve (BPHV) degeneration, characterized by extracellular matrix deterioration, remodeling, and calcification, is an important clinical problem accounting for thousands of surgeries annually. Here we report for the first time, in a series of in vitro accelerated fatigue studies (5-500 million cycles) with glutaraldehyde fixed porcine aortic valve bioprostheses, that the mechanical function of cardiac valve cusps caused progressive damage to the molecular structure of type I collagen as assessed by Fourier transform IR spectroscopy (FTIR). The cyclic fatigue caused a progressive loss of helicity of the bioprosthetic cuspal collagen, which was evident from FTIR spectral changes in the amide I carbonyl stretching region. Furthermore, cardiac valve fatigue in these studies also led to loss of glycosaminoglycans (GAGs) from the cuspal extracellular matrix. The GAG levels in glutaraldehyde crosslinked porcine aortic valve cusps were 65.2 +/- 8.66 microg uronic acid/10 mg of dry weight for control and 7.91 +/- 1.1 microg uronic acid/10 mg of dry weight for 10-300 million cycled cusps. Together, these molecular changes contribute to a significant gradual decrease in cuspal bending strength as documented in a biomechanical bending assay measuring three point deformation. We conclude that fatigue-induced damage to type I collagen and loss of GAGs are major contributing factors to material degeneration in bioprosthetic cardiac valve deterioration.     

Effect of lipopolysaccharide on proteoglycan synthesis by adult human gingival fibroblasts in vitro.

The effect of lipopolysaccharide preparations from Salmonella enteritidis, Bacteroides gingivalis, and Actinobacillus actinomycetemcomitans on human gingival fibroblasts was studied. Lipopolysaccharide from all sources inhibited fibroblast proliferation in the concentration range of 0.5 to 50 micrograms/ml, with the lipopolysaccharide from A. actinomycetemcomitans having the strongest inhibitory effect. Assessment of the effect of lipopolysaccharide on gingival fibroblast metabolism indicated both total protein and proteoglycan synthesis to be inhibited with increasing concentrations of lipopolysaccharide. As for the antiproliferative effect, lipopolysaccharide from A. actinomycetemcomitans had the greatest inhibitory effect on cell synthetic activity. This inhibitory effect was determined by pulse-chase experiments to be a true depression in synthesis. Furthermore, the effect was independent of lipopolysaccharide-induced changes in cell proliferation and prostaglandin synthesis. This study confirmed the toxic effect of lipopolysaccharide on fibroblasts and, in particular, indicated that various lipopolysaccharide preparations vary in their potency to influence cell proliferation and extracellular matrix synthesis.     

The closing velocity of mechanical heart valve leaflets

The closing motion of the occluder leaflets in bileaflet type mechanical heart valves (MHV) was monitored with a laser sweeping technique. The angular displacements of the leaflets were registered with precision of 0.2 μs steps. Experimental measurements were made using five 29 mm Edwards-Duromedics™ including three original specification (EDOS) and two modified specification (EDMS), and two 29 mm St Jude Medical® MHVs. The testing valve was installed in the mitral position of a physiologic pulsatile mock circulatory flow loop using water-glycerine solution as the testing fluid. Each valve was tested by: (1) direct mounting the valve on metal washers, and (2) mounting the valve with its sewing ring. Experiments were carried out at pulse rates of 70, 90, and 120 beats min⁻¹, with the corresponding cardiac output of 5, 6, and 7.5 litres min⁻¹, and maximum left ventricular pressure gradients ( ) of 1,800, 3,000 and 5,600 mm Hg s⁻¹, respectively. The maximum leaflet closing velocity of each of the tested valve types are presented. The difference in leaflet closing movements between the direct rigid mounting and the sewing ring mounting are discussed. The details of the laser sweeping technique are presented.