INTRODUCTION 14 1 2– 5 9 15– 17 3 9 15 4 5 16 2 3 17 19 4 5 10 7 By measuring the cell forces under various cyclic stretch amplitudes and frequencies, we can better understand the relationship between the applied strains and the cells’ mechanical response. Then we can model this behavior and be able to predict cellular responses to dynamic physiologic and pathologic loading conditions. METHODS Tissue Ring Fabrication 20 1 FIGURE 1. (A) (B) (C) (D) 4  FIGURE 2. Illustration of peak force versus time. The gray curve is sample data obtained for 10 stretch cycles. The black line plots the peak force for each cycle vs. time. This peak force line is used for many of the following figures.  Mechanical Tests 1 20 Effect of Cyclic Stretch Amplitude 2 21 FIGURE 3. Cell force calculation. Matrix force was measured by adding 2μM CytoD to rings prior to initiation of cyclic stretch. Cell force is calculated by taking the normalized matrix force (see Eq. 1) and subtracting it from the tissue force.  F C0 t F C12 t) P C0 t P C12 t 1 \documentclass[12pt]{minimal}
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\begin{document}$$F_{C0}^{\left( {sc} \right)} \left( t \right) = F_{C0} \left( t \right)\frac{{P_{C12} \left( {14\,{\rm hr}} \right)}}{{P_{C0} \left( {14\,{\rm hr}} \right)}}$$\end{document} 3 2 \documentclass[12pt]{minimal}
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\begin{document}$$F_{{\rm cell}} \left( t \right) = F_{C12} \left( t \right) - F_{CO}^{\left( {sc} \right)} \left( t \right)$$\end{document} Single Step Stretch and Hold Effect To compare the effect of a single stretch to cyclic stretching, a step stretch (stretch to a given stretch magnitude in less than 0.1 s) of 5%, 10%, or 20% was applied and the rings were held at this stretched length for 8 h while the force was monitored. The tissue was then returned to its baseline length and the force was monitored for an additional 6 h. In another set of experiments, CytoD was added 3 h before or 12 h after either a 10% or 20% step stretch. Effect of Cyclic Stretch Frequency A similar procedure was followed to test frequency effects. Four rings were tested at each of the three days with each ring stretched at either 0.25 Hz, 0.1 Hz, 0.01 Hz, or 0.001 Hz at 10% stretch amplitude. The current tester’s upper frequency limit is 0.25 Hz and thus unable to stretch at higher frequencies that are closer to physiologic values. CytoD was added either 3 h before or 12 h after initiation of stretch to separate out the cell and matrix components as a result of different frequencies of cyclic stretch. Cell Number After the completion of testing, some rings were washed in phosphate buffered saline (PBS) and then placed in 1 ml lysis buffer (0.1% sodium dodecyl sulfate (SDS) in PBS). The samples were sonicated until the rings completely disintegrated to release the DNA from the cells. Finally, 30 μl of sample was placed in 3 ml of Hoechst working solution (30 nM Hoechst 33258 dye (Sigma, St. Louis, MO) in PBS). The fluorescence was then read by a spectrophotometer and cell number determined from a standard curve obtained from samples with known numbers of cells. Immunohistochemistry Rings were removed from spacers after 2, 4, or 8 days static incubation and immediately placed in a solution of 4% paraformaldehyde for 30 min, washed with PBS and then permeabilized for 45 min in 0.2% Triton X-100 in PBS. This was followed by 1 h incubation in a blocking solution (2% normal goat serum and 0.02% sodium azide in PBS). After blocking, the rings were incubated overnight at 4°C in blocking solution containing TRITC phalloidin. Finally, the rings were washed again in PBS and mounted for viewing on a confocal microscope (Zeiss Confocor). RESULTS Ring Width 4 FIGURE 4. (A) (B)  Cell Number 4 Amplitude Effect 5 5 5 5 5 n 5 Tissue Force 6 5 FIGURE 5. Peak tissue and matrix forces for different cyclic stretch amplitudes at 0.1 Hz. (A) Peak tissue force of the first cyclic stretch after 2, 4, or 8 days static incubation. Peak tissue force increases with cyclic stretch amplitude and incubation time. (B) Peak tissue force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. At day 8 the peak tissue force is similar regardless of cyclic stretch amplitude. (C) Peak matrix force of the first stretch after 2, 4, or 8 days static incubation. Peak matrix force increases with cyclic stretch amplitude. (D) Peak matrix force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. Higher amplitudes result in higher peak forces. (E) Number of rings averaged for data shown in Figures 5-8 at each amplitude and static incubation day. (F) Individual measurements used in panel B [Peak tissue force after 12 h, 8 days of incubation]. This shows that the trend within an experiment, i.e. rings fabricated on a single day, is consistent but the experiment to experiment variation in forces is large. Y-axis scales are different in each panel to emphasize the trends with amplitude and incubation time.  FIGURE 6. (A) (B) (C)  6 6 6 FIGURE 7. (A) (B) (C)  FIGURE 8. (A) (B) (C)  FIGURE 9. Response to step stretch and hold after 8 days of static incubation. The tissue, matrix, and cell forces are shown after a step stretch of either 10% or 20%. The tissue is held at the stretched length for 12 h. The tissue and matrix force for a 20% step and hold is higher than that for a 10% step and hold. The cell force is identical for 10% and 20%.  Matrix Force 7 7 7 Cell Force 8 8 8 Single Step Stretch & Hold Effect 9 9 9 Rings that did not have CytoD added before or during the step stretch were returned to their baseline lengths after being held at the stretched length for 8 h. The force dropped to zero but then began to recover with lower magnitude step stretches increasing their forces faster than higher magnitude step stretches. The recovered forces approached their baseline forces prior to stretch. After 6 h of monitoring force at baseline, rings stretched to 5% magnitude and held recovered more of their baseline force (84%) than rings stretched to 20% magnitude (42%). The force decreases more rapidly in the step stretch and hold than the peak force drops in cyclic stretching at the same amplitude. Frequency Effect 10 10 10 10 FIGURE 10. (A) (B) (C) (D) (E) (F)  Tissue Force 11 FIGURE 11. (A) (B) (C)  Matrix Force 12 12 12 FIGURE 12. (A) (B) (C)  Cell Force 13 13 13 FIGURE 13. (A) (B) (C)  Immunohistochemistry 14 14 14 14 14 FIGURE 14. (A) (B) (C) (D) (E) (F)  DISCUSSION 18 While the initial extracellular matrix consists entirely of collagen it is reasonable to assume that over 8 days of incubation time that additional cell-contributed matrix may be produced. We have incubated rings for 20 days prior to cyclic stretching and found that the matrix force-strain curves are similar to those after 8 days of incubation. Peak forces increase only a small amount at 5% or 10% stretch amplitude after 14 h of cyclic stretch at 0.1 Hz. These results suggest that the matrix mechanics do not change substantially even in the presence of cells that can produce additional matrix. 9 9 6 Since it is the cells that are responding to the mechanical conditioning it is important to realize that while the cells are undergoing a higher strain they are not undergoing a higher stress. So if the cells respond to stress, higher stretch amplitudes will not increase this response. Since both the cells and the tissues at different stretch amplitudes maintain similar stresses, it is reasonable to assume the cells are responding to maintain a certain range of mechanical stresses. 11 Unlike the cells that respond to increased stretch amplitude by decreasing their stiffness, the matrix force-strain curves shift so that the matrix contributes only when the strain is within 3%–7% of the maximum value with the matrix contributing over a slightly larger range of strains at higher stretch amplitudes. This response of the matrix does not appear to depend upon the cells having an actin cytoskeleton, since we found a similar response when cytoD is added either before stretch is initiated or after 12 h of stretch. After 12 h of cyclic stretch, the peak matrix force is only slightly higher with increased stretch amplitude. With different frequencies of cyclic stretch, the peak matrix force is similar but the matrix contributes over a larger range of strains at lower frequencies. For a 10% stretch, the matrix contributes to the last 6% of the stretch at 0.001 Hz while contributing only to the last 4% at 0.25 Hz. The higher baseline tissue and cell forces seen at lower frequencies (0.001 Hz and 0.01 Hz) are consistent with a simple viscoelastic model because the tissues and cells have more time to recover before being stretched again. The same is seen in the lower amplitudes at 0.1 Hz (5% and 10%) which are able to maintain a baseline force. These correspond to the lowest strain rates and may allow for recovery. For a linear viscoelastic material undergoing continuous cyclic stretch, both higher stretch amplitudes and higher strain rates will lead to higher peak forces. For cyclic stretch at constant frequency, the highest stretch amplitude also corresponds to the highest strain rate. However, we observed that the peak cell force decreased as the amplitude (and the strain rate) increased. We also note that the decrease in peak cell force is a function of both stretch amplitude and strain rate because similar strain rates (5%/s) at different amplitudes (e.g. 10% @ 0.25 Hz and 25% @ 0.1 Hz) show different tissue, matrix and cell responses In the case of the matrix at 25% @ 0.1 Hz, there is no positive matrix force until the strain increases beyond 10%; whereas there is matrix force below 10% strain during cyclic stretch at 10% @ 0.25 Hz. 8 13 3 12 3 3 3 3 In addition, these results have important implications for the tissue engineering field, especially for those tissues that undergo continuous deformations such as in the cardiovascular system. By isolating the cell contribution to tissue force, we are able to provide a better understanding of the mechanical environment of the cell and aid in designing mechanical conditioning protocols that improve tissue properties. For example, the peak cell force is not constant during cyclic stretch and thus cell stimulation varies with time. Furthermore, if cell response is dependent on cell stress or force, increasing the amplitude of conditioning will not increase and may decrease the applied cell force. 4 5 9 16 CONCLUSIONS We have shown that bioartificial tissue constructs consisting of a single cell type and matrix component are a useful model system in which to explore tissue, matrix, and cell mechanics. The cell response to cyclic stretch is complex and depends on both the amplitude and frequency of cyclic stretch. The change in cell stiffness with amplitude behavior does not fit current cellular mechanics models. Future investigations may explore the microstructural basis for this behavior.