Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro.

Cells in the body are constantly subjected to cyclic mechanical deformation involving tension, compression, or shear strain or all three. A mechanical loading system which deforms cultured cells in vitro was analyzed in order to quantify the deformation or strain to which the cells are subjected. The dynamic system utilizes vacuum pressure to deform a circular silicone rubber substrate on which cells are cultured. These thick circular growth surfaces or plates are formed in the bottoms of the wells of 6-well culture plates. An axisymmetric model was formulated and analyzed using rectangular hyperelastic elements in a finite element analysis (FEA) software package. The thick circular plate has some disadvantages such as difficulty in observing cells and a nonhomogeneous strain profile which is maximum at the periphery and minimal at the center. A thinner circular surface (a thin plate) was also investigated in order to provide a more homogeneous strain profile. The radial strain on the thick circular plate, as determined by FEA, was nonlinear with a peak strain value of 0.30 (vacuum pressure of 22 kPa) about three-quarters of the distance from the center to the edge. In contrast, the radial strain of the thin circular plate was moderately constant across the surface. The circumferential strain for both of these models was less than the radial strain except for the center where they are equal. Avian tendon cells were cultured on the surface of a thick plate and exposed to cyclic strains for 24 h at a rate of 0.17 Hz and observed for cellular alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of endothelial cell phenotype by cyclic stretch: inhibition of collagen production.

Endothelial cells (EC) are constantly subjected to pulsatile stretch in vivo, but most studies of EC are performed under stationary tissue culture conditions. The aim of this study was to examine the effect of repetitive mechanical stretching on EC collagen production. Bovine EC were seeded in 35-mm flexible-bottom culture wells, allowed to attach for 24 hr, and then subjected to up to 24% elongation cyclic deformation, 3 cycles/min, 10 sec of elongation alternating with 10 sec of relaxation, for 5 days. Twenty-four hours prior to harvesting, serum-free media containing 50 microCi [3H]proline (PRO), an amino acid hydroxylated (OH) in collagen, and 50 micrograms/ml ascorbate were added per well. On Days 1, 3, and 5, the media and cells were collected, precipitated with TCA, sedimented, lyophilized, and analyzed by HPLC for OH-PRO and PRO. The results of this study indicate that EC production of collagen is inhibited with repetitive deformation. Since previous studies have shown that EC proliferation is increased after 3 cycles/min stretching, this supports the theory that collagen gene expression varies inversely with the proliferative state.

Differences in secretion of prostacyclin by venous and arterial endothelial cells grown in vitro in a static versus a mechanically active environment.

The objective of this study was to compare the effect of cyclic tension on prostacyclin secretion by venous and arterial endothelial cells. Early passage endothelial cells from bovine aortas and venae cavae were subjected to cyclic deformation (up to 17% elongation and 60 cycles/min). On posttreatment days 3 and 5 a radioimmunoassay was used to assess supernatant fluids from the endothelial cells for prostacyclin activity. The results indicate that in vitro (1) under static or control conditions, venous endothelial cells secrete significantly more prostacyclin (an increase of 1.5- and 4.8-fold on days 3 and 5, respectively) than do arterial endothelial cells isolated from the same animal and (2) prostacyclin secretion by mechanically deformed venous and arterial endothelial cells was significantly less than static control cultures on days 3 and 5. However, prostaglandin I2 secretion remained at higher levels in cyclically deformed venous endothelial cells than in cyclically deformed arterial endothelial cells. These data suggest that endothelial cells from the vena cava have a greater capacity for prostacyclin secretion than have their aortic counterparts. If these observations are maintained in vivo, greater prostacyclin secretion by venous endothelial cells could represent a homeostatic mechanism aimed at reducing thrombus formation in low-velocity areas of the vasculature.

Osteoblasts increase their rate of division and align in response to cyclic, mechanical tension in vitro.

Bone adapts to physical deformation in vivo, yet the mechanism of the adaptive process remains unknown. One reason for this perplexity has been the difficulty in examining the effects of a well-defined deformation regimen on individual bone cells. With the utilization of novel, flexible-bottomed cell culture plates, one can study the effects of cyclic strain on the morphologic and biochemical adaptations of individual osteoblasts in vitro. Avian, calvarial osteoblast-like cells, from passes 2-5, responded to cyclic strain, by increasing their rates of DNA synthesis and cell division during the first 72 h after initiation of a continuous deformation regimen comprised of 3 cycles per min of 0-24% elongation. In addition, within hours after initiation of the deformation regimen, cells oriented 90 degrees to the applied strain field at the periphery of the culture plate in the region of maximum strain and elongation.

Tendon synovial cells secrete fibronectin in vivo and in vitro.

The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of 35S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.

Cell populations of tendon: a simplified method for isolation of synovial cells and internal fibroblasts: confirmation of origin and biologic properties.

Tendons transmit the force of muscle contraction to bone to effect limb movement. Special structural and biological properties of tendon have developed to facilitate force transmission. The tendon has a complex organization of cells surrounding the collagen bundles inside tendon as well as at the tendon surface. Internal cells may act to maintain the bulk of the collagen in tendon. External cells in the epitenon may provide lubrication for tendon gliding. To develop better understanding of these processes and the roles the cell populations play, we isolated cells from the surface and interior of tendon and studied them in vitro. Flexor tendons from 8-week-old white Leghorn chickens were separated into two distinct cell populations: the outer synovial cells and the fibroblasts more internal in tendon. These cell populations were discernible by their locations in the intact tendon, determined by sequential enzymatic and physical release from their substrata. Initially, some cells eluted in Hanks' salt solution (HSS) (population 1); then synovial cells were released after a 2-min treatment with 0.5% collagenase (population 2). Next, a population of synovial cells was released in high yield by treatment with 0.25% trypsin (step III, population 3). Step III, population 3 cells were used as synovial cells (SCs). Next, a population of SCs and fibroblasts were released by scraping with a rubber policeman (population 4). Subsequently, fibroblasts were released after incubation with 0.5% collagenase (population 5). A more direct procedure (procedure 2) to isolate the synovial and internal tendon cells involved treatment in 0.5% collagenase followed by sedimentation at 900 g. Cells that sedimented were largely fibroblasts, whereas the cells that remained at the top of the tube were largely SCs. Cells designated as SCs, isolated by procedure 2, most likely contained surface cells from epitenon and internal interfascicular cells from endotenon and paratenon. Surface tendon cells separated by sequential enzymatic and physical release from their substrata (by procedure 1) had all the following characteristics: distinct subpopulations of cells based on morphology; presence of cytoplasmic, lipid-containing vesicles; decreased sensitivity to trypsin; and reduced generation time as compared with that of internal fibroblasts. Conversely, the internal fibroblasts (IFs) appeared to represent a more uniform population based on morphological characteristics.

High-performance liquid chromatographic quantitation of rhodamines 123 and 110 from tissues and cultured cells.

Rhodamine 123 is a fluorescent vital dye which has potential for therapeutic use in cancer treatment. The dye concentrates in mitochondria of normal and neoplastic cells but accumulates in and is toxic to neoplastic cells. When dye-treated cells are irradiated with blue laser light at 514 nm, mitochondrial injury or cell death results. Rhodamine concentration in cultured cells and tumor tissue was quantitated to correlate cell or tumor death with drug dose. A reversed-phase separation of rhodamine 123 was accomplished using a gradient of 0.05 M phosphate buffer pH 2.85 (mobile phase A) and acetonitrile (mobile phase B), 10-80% B in 15 min with a DuPont Golden Series C8 column. Effluent was monitored with a fluorescence detector at 295 nm excitation and 520 nm emission. Stock rhodamine 123 contained approximately 6-8% of rhodamine 110, the parent compound, which eluted at 9.8 min whereas rhodamine 123 eluted at 11.7 min. Structural verification of both compounds by field desorption mass spectrometry was performed. This is the first report of the chemical separation and quantitation of rhodamine 123 from cultured tumor cells or tumor tissue.

Effects of solutions used for storage of size-exclusion columns on subsequent chromatography of peptides and proteins.

The effects of storage of size-exclusion column packing materials in methanolic or azide-water solutions on subsequent separations were tested. Three commercially available columns were used in these studies; the Toyo-Soda Bio-Sil TSK 125, Bio-Sil TSK 250 and the DuPont Bio-Series GF-250. Upon initial chromatography, all three columns bound up to 760 micrograms of cytochrome c tryptic peptides. Sample binding to packing material is probably a function of the positively charged basic groups on peptides or proteins interacting with silanol groups. The larger the peptide, the less the opportunity for silanol-charged group interaction, hence, less binding. Initial samples introduced to a new column occupy the binding sites. Equilibration with neat methanol removes the bound protein revealing sites which bind sample. After absorption of peptides to binding sites on the packing material, storage in neat methanol regenerates the binding sites. Storage in 10% methanol diminished the binding phenomenon, but storage in azide-water reduced binding to a range below detection at the microgram level. Our recommendation to users of size-exclusion chromatographic columns is that one satisfy the absorption capacity of a new column by injecting a sufficient quantity of a basic peptide standard or other convenient sample to reduce available binding sites before using the column for important separations. Store columns in azide-water or 10% methanol to prevent the regeneration of exposed silanol groups.

Comparison of reversed-phase columns for the separation of tryptic peptides by gradient elution. Correlation of experimental results and model prediction.

A total of 10 reversed-phase columns obtained from different suppliers were evaluated for their ability to separate typical peptide mixtures: tryptic digests of cytochrome c, lysozyme and collagen. Each column was tested in a standard gradient elution using phosphate buffer (pH 2.85) as the A-solvent and acetonitrile as the B-solvent. Some differences in band spacing for the various peptides were observed from column to column. More important differences were observed in the ability of these various columns to provide narrow peaks and good resolution, as measured by column peak capacity. These differences in column peak capacity were related to differences in column dimensions and particle size; it also appeared that C8 bonded phases were somewhat more efficient than were C18 phases. The recovery of cytochrome c digest from four of the columns was also assessed. Summed peak areas were proportional to sample size, as the latter was varied from 1 to 100 micrograms. The apparent recovery from each column did not vary by more than +/- 5%. From this it was concluded that these columns gave essentially quantitative recovery for this particular sample. Limited data on column life were obtained for some of these columns.