The Quantification of Shape Change in MC3T3 Cells Grown on Smooth and Microgrooved Silicone SubstratesMaura George, James H-C. Wang, Ph.D.
Musculoskeletal Research Center
Department of Orthopaedic Surgery
University of Pittsburgh Medical Center, PA
IntroductionPrevious studies have observed an interesting occurrence upon growing fibroblasts and osteoblasts on substrates with a microgrooved surface; the cells orient themselves in the direction of the microgrooves. In addition, several studies have reported changes in the shape of cells due to the microgrooves (Wang et al., 2000).
The cells grown on microgrooved, as opposed to smooth, substrates are generally more elongated and tend to conform to the shape of the microgrooves. MC3T3 cells have also been shown to manifest this phenomenon (Wang et al., 2000). While extensive research has reported such findings, the evidence has been mainly qualitative with little effort to objectively measure the shape change. The objective of this study was to quantify the shape of cells on smooth and microgrooved surfaces.
MC3T3-E1 osteoblastic cells were used, as they well represent osteoblasts and can undergo many passages without differentiation.
Methods and Materials
Plastic 6-well plates (Becton Dickinson Labware) were used to grow the cells. Thin, smooth silicone membranes were purchased from Specialty Manufacturing. Pronectin-F was obtained from Protein Polymer Technologies. MC3T3-E1 osteoblasts, derived from mouse calvaria, were used in experiments.
Cell cultureThe cells were grown in a-MEM with 10% fetal bovine serum, 1% penicillin/streptomycin, b-glycerol phosphate (5mM), and ascorbic acid (25 ug/ml) and incubated at 37°C. The atmosphere of the incubator was humid and composed of 95% air and 5% carbon dioxide
Preparation of substratesMicrogrooved silicone pieces were obtained by a molding against wafers, which were made by lithographic and reactive etching method. Smooth silicone circles and microgrooved silicone rectangles of equal area were cut. These pieces were glued into multi-well plates using silicone glue and exposed to UV radiation for several hours. A solution of Pronectin-F (10ug/mL) was used to coat the silicone for 10 min, after which the pieces were washed twice with phosphate buffered saline (PBS).
Photographing of cellsThe cells, grown to confluence in a 100 mm petri dish, were then detached using Trypsin (0.25%) and transferred to 6-well plates containing the smooth and microgrooved silicone pieces. After the cells were incubated for three days, they were photographed with Kodak 100 slide film under a phase contrast microscope.
Measurement of cell perimeter and areaThe resulting slides were scanned into the computer and digitized. The cells perimeter and area were measured with Scion Image. These values were entered into Microsoft Excel to calculate the shape index. The shape indices of the smooth and microgrooved cells were calculated from the following formula:
Table 1: Shape indices for several common shapes
StatisticsFor statistical analysis of the data, an unpaired t-test was used, with a significance level set at 0.05.
ResultsAn example of slides taken of cells grown on microgrooved (A) and smooth substrates (B) are shown in Figure 1.
Figure 1: MC3T3 cells on (A) smooth and (B) microgrooved substrates
Figure 2 shows tendon fibroblasts embedded in a collagen fiber section. These cells naturally align in a similar manner to the cells on the microgrooved substrate.
Figure 2: H&E staining of a tendon fibroblast section
Although Figure 1 (B) is from an in vitro sample, a similar orientation may be seen when studying in vivo cells, such as those in Figure 2. The results of the calculated shape factors are shown in Figure 3. The tendon fibroblast cells are shown for comparison.
DiscussionThis study showed the shape of MC3T3 cells grown on a smooth silicone substrate to be significantly different from that of the same cells grown on a microgrooved silicone substrate. Also, the tendon fibroblasts in vivo had shapes that were similar to the osteoblastic MC3T3 cells grown on the microgrooved surfaces.
The change in shape between the cells on smooth and microgrooved substrates most likely results from contact guidance (Wang et al., 2000) provided by the grooves. This guidance directs the actin cytoskeleton to grow along the grooves, as this is the path of least mechanical resistance.
The differences in shape and alignment may result in differences in the proliferation and differentiation of the osteoblasts, as a cell's shape influences many of its functions (Wang 2000). These functions include the synthesis of proteins such as pro-collagen. Further studies may therefore include measurements of the relative proliferation and the difference in protein synthesis between MC3T3 cells grown on smooth and microgrooved silicone substrates.
To conclude, this study has quantifiably shown the change of shape that occurs in osteoblastic cells when grown on microgrooved substrates. The cells become more linear and orient themselves in the direction of the grooves.
References1. Quarles et al., J. of Bone and Mineral Research 7:683-692, 1992.
2. Wang. J. theor. Biol. 202:33-41, 2000.
3. Wang et al., J. of Biomechanics 33:729-735, 2000.
AcknowledgmentsThe author thanks Dr. Savio L-Y. Woo for the opportunity of and guidance in this project and the MSRC for their help and support.