[Preliminary study on interactions between endothelial cells and domains with different protein concentrations on the same substrate].
OBJECTIVE
To investigate the biological response and chemotaxis of endothelial cells on template materials with different protein concentrations on the same surface, to provide the evidence for deep understanding of chemical induced cell motility.


METHODS
Microcontact printing technique was employed to fabricate template materials with four different concentrations of collagen (50, 100, 200, 300 microg/mL) on the same substrate. Scanning electron microscopy was employed to characterize the quality of polydimethylsiloxane (PDMS) stamp. Confocal laser scanning microscopy (CLSM) was utilized to characterize the absorption of different concentrations of FITC conjugated collagen (50, 100, 200, 300 microg/mL) on the substrates surfaces. Software was used to analyze the fluorescence intensity of adsorbed protein on the substrates. Albumin was then used to block the substrates for cell culture of human umbilical vein endothelial cells (hUVEC). Substrates with no collagen adsorption were used as control samples. The influence of different concentrations of collagen on the proliferation of hUVEC was investigated via MTT assay at 6, 24, 48 and 72 hours of culture. The cytoskeletal structures of cells were characterized by CLSM. The cell's migration speed and absolute displacement were measured by path measurement of single cell after 24 hours of culture.


RESULTS
Fabricated PDMS stamps with complete pattern were flat. Template substrates were fully covered with evenly distributed collagen protein. The fluorescence intensities were 38.51 +/- 1.63, 55.21 +/- 3.88, 73.17 +/- 3.59, and 80.95 +/- 1.12 in adsorbed FTIC conjugated collagen with 50, 100, 200 and 300 microg/mL, respectively. Endothelial cells spread better on various substrates coated with collagen than those of control samples. The proliferation of endothelial cells on collagen coated substrates was significantly higher than that of control group (P < 0.05). With collagen concentration increasing from 50 microg/mL to 300 microg/mL, the proliferation abilities and absolute displacements of endothelial cells significantly increased (P < 0.05). Except for the group with 300 microg/mL, the migration speed of endothelial cells on collagen coated substrates was significantly lower (P < 0.05) than that of control group. However, the migration speed of endothelial cells on collagen coated substrates significantly increased (P < 0.05) along with collagen concentration increasing from 50 microg/mL to 300 microg/mL.


CONCLUSIONS
It is feasible to acquire domains with different protein concentrations on the same substrate using microcontact printing technique for investigating cell's chemotaxis.
Chen
Chong
C
Bioengineering College, Chongqing University, Chongqing, 400044, P.R. China.
Cai
Kaiyong
K
Schroeter
Siegmund
S
D018929:Cell Culture Techniques / Q000379:methods; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D018919:Neovascularization, Physiologic; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D003094:Collagen