No abstract

Many types of cells, when grown on the surface of a cyclically stretched substrate, align away from the stretch direction. Although cell alignment has been described as an avoidance response to stretch, the specific deformation signal that causes a cell population to become aligned has not been identified. Planar surface deformation is characterized by three strains: two normal strains describe the length changes of two initially perpendicular lines and one shear strain describes the change in the angle between the two lines. The present study was designed to determine which, if any, of the three strains was the signal for cell alignment. Human fibroblasts and osteoblasts were grown in deformable, rectangular, silicone culture dishes coated with ProNectin, a biosynthetic polymer containing the RGD ligand of fibronectin. 24 h after plating the cells, the dishes were cyclically stretched at 1 Hz to peak dish stretches of 0% (control), 4"%, 8%. and 12% After 24 h of stretching, the cells were fixed, stained, and their orientations measured. The cell orientation distribution was determined by calculating the percent of cells whose orientation was within each of eighteen 5" angular intervals. We found that the alignment response was primarily driven by the substrate strain which tended to lengthen the cell (axial strain). We also found that for each cell type there was an axial strain limit above which few cells were found. The axial strain limit for fibroblasts, 4.2&0.4% (mean & 95% confidence), was lower than for osteoblasts, 6.4 * O.6' Li . We suggest that the fibroblasts are more responsive to stretch because of their more highly developed actin cytoskeleton.

We developed an experimental system to stimulate cell cultures by uniform and cyclic biaxial strain of the cell culture surface. The studies reported here were designed to determine the uniformity of the strain distribution, the suitability of the surface for the growth of human osteoblasts, and the effects of strain magnitude on cell proliferation and alkaline phosphatase (AP) activity. Subconfluent cell cultures were grown in rectangular silicone dishes that were stretched cyclically (1 Hz) in the long axis by an electromechanical apparatus that controlled peak stretch and cycle frequency. We applied cyclic strains (1.0, 2.4, 5.3, and 8.8% surface strains) for 15 minutes per day on 3 consecutive days. Phase contrast microscopy confirmed the transfer of dish surface strain to the cells. Stretching of the dish resulted in a homogeneous strain distribution that deviated approximately 0.05% from the applied strain. In comparison with plastic dishes, there was a 20% reduction of cell proliferation on the silicone substrate whereas morphology, AP activity, and total protein content of the cells were similar. The proliferation of human osteoblasts was increased significantly (16.4-100%) by 1% strains, although higher strain magnitudes had lesser (nonsignificant) effects or decreased the mitotic activity of the cells. AP and lactate dehydrogenase activities were not influenced significantly by cyclic strains. This study demonstrates that the cell stretching system is suitable for the investigation of the effects of well defined cyclic strains.

Study Design Responses of mesenchymal stem cells (MSCs) from 2 age groups was analyzed under chemical conditions representative of the intervertebral disc (IVD) (low glucose levels, acidic pH, high osmolarity, and combined conditions). Objective To determine the microenvironmental conditions of the IVD that are critical for MSC-based tissue repair and to determine whether MSCs from different age groups respond differently. Summary of Background Data MSCs offer promise for IVD repair, but their potential is limited by the harsh chemical microenvironment in which they must survive. Methods MSCs were isolated from bone marrow from mature (4–5 month old) and young (1 month old) rats and cultured in monolayer under IVD-like glucose, osmolarity, and pH conditions as well as under a combination of these conditions and under standard media conditions for 2 weeks. The response of MSCs was examined by measuring gene expression (real-time RT-PCR), proliferation (MTT assay), and viability (fluorescence staining). Results Culturing under IVD-like glucose conditions (1.0 mg/mL glucose) stimulated aggrecan and collagen-1 expression and caused a small increase in proliferation. In contrast, IVD-like osmolarity (485 mOsm) and pH (pH = 6.8) conditions strongly decreased proliferation and expression of matrix proteins, with more pronounced effects for osmolarity. Combining these 3 conditions also resulted in decreased proliferation, and gene expression of matrix proteins, demonstrating that osmolarity and pH dominated the effects of glucose. Both age groups showed a similar response pattern to the disc microenvironment. Conclusion IVD repair using MSCs requires increased knowledge of MSC response to the chemical microenvironment. IVD-like low glucose enhanced matrix biosynthesis and maintained cell proliferation whereas IVD-like high osmolarity and low pH conditions were critical factors that reduced biosynthesis and proliferation of young and mature MSCs. Since osmolarity decreases and acidity increases during degeneration, we speculate that pH may be the major limitation for MSC-based IVD repair.

Intervertebral discs (IVD) have a higher extracellular osmolarity than most other tissues; moreover their osmolarity changes by around 25% during each diurnal cycle. In this study, changes in aggrecan, collagen I and collagen II expression of IVD cells were examined after exposure to osmotic environment alterations or mechanical stimulation under different osmotic conditions. Human and bovine IVD cells seeded in three-dimensional (3D) collagen type I matrices were cultured under hypo-osmotic (300 mOsm), iso-osmotic (400 mOsm), or hyperosmotic (500 mOsm) conditions. Osmolarity-induced changes in gene expression of IVD cells were measured after 5 days. Loadinduced changes in gene expression under the different osmotic conditions were measured after application of hydrostatic pressure (0.25 MPa, 0.1 Hz, 30 min) or cyclic strain (4%, 1 Hz, 24 h). The results showed that IVD cells respond strongly to changes in the osmotic environment by altering mRNA expression. Human cells cultured over 5 days increased expression of aggrecan and collagen II in both nucleus and annulus cells under increasing osmolarity. In contrast, collagen I expression was inhibited at high osmolarity in both cell types. Mechanically induced alterations in gene expression appear to have only modest effects on matrix protein expression, but the same stimulus partly resulted in an inhibition or stimulation of gene expression, depending on the osmotic conditions. This study showed that the osmotic environment does not only have an appreciable effect on gene expression but also affects responses to mechanical stimuli. This suggests that the osmotic conditions cannot be ignored when examining physiological and pathological behavior of IVD cells.