Delivery of growth factors to target cells is an important subject in tissue engineering. Towards that end, we have developed a growth factor-tethered extracellular matrix (ECM). Here, basic fibroblast growth factor (bFGF) was tethered to extracellular matrix noncovalently. The designed ECM was comprised of 12 repeats of the APGVGV peptide motif derived from elastin as a stable structural unit and included the well-known cell adhesive RGD peptide as an active functional unit. To bind bFGF to the ECM, an acidic amino acid-rich sequence was introduced at the C-terminus of the ECM protein. It consisted of 5 repeats of 4 aspartic acids and a serine, DDDDS. bFGF has a highly basic amino acid domain. Therefore, bFGF was tethered to the ECM protein by electrostatic interaction. Cells cultured on bFGF-tethered ECM were well attached to the ECM and induced proliferation without addition of soluble bFGF.
Growth factors are important for regulating a variety of cellular processes, and they are indispensable for tissue engineering. To increase the local concentration of growth factors, several techniques for delivering growth factors have been investigated [
A strategy to immobilize growth factors to ECMs has advantages compared to the addition of soluble growth factors. When soluble growth factors are added to cells, it is difficult to control their local concentration due to diffusion, cell uptake, and degradation [
Basic fibroblast growth factor (bFGF) is a commonly used growth factor for tissue engineering because of its wide variety of functions. For example, it is a stimulator of proliferation, differentiation, and migration of multiple cell types [
In this study, bFGF-tethered ECM was developed for the purpose of delivering growth factors to cells. For tethering bFGF, a polyaspartic acid domain (D20) was introduced to our designed artificial ECM, ERE, which consists of 12 repeats of the Ala-Pro-Gly-Val-Gly-Val (APGVGV) motif derived from elastin as a stable structural unit. The repeated APGVGV sequence is highly hydrophobic, allowing ERE to adsorb well onto the hydrophobic surface of the dish. It also included the well-known cell adhesive RGD sequence as an active functional unit [
Schematic drawing of bFGF-tethered designed ECM through electrostatic interaction.
The plasmid pET-ERE constructed in our laboratory was digested with
The constructed plasmid, pET-His-ERE-(D4S)5, was transfected into
The murine fibroblast cell line C3H10T1/2, obtained from the Riken Cell Bank, was grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100
Solutions of ERE-D20 and ERE were added to 96-well suspension culture plates (Sumilon, MS-8096R) in varied concentrations and incubated for 2 h at 37°C with shaking. Plates were washed with PBS-T (PBS including 0.05% Tween 20) followed by blocking with Blocking One (Nacalai Tesque, Inc.) overnight at 4°C. After washing with PBS-T, anti-poly-histidine antibody (Sigma-Aldrich) was added to the plate and incubated for 1 h at 37°C followed by washing with PBS-T again. Then, anti-mouse IgG peroxidase conjugate (Sigma-Aldrich) was added and incubated for 1 h at 37°C. After washing with PBS-T, TMB peroxidase substrate (KPL, Inc.) was added to the plate. Finally, 1 M HCl was added to stop the reaction and the absorbance at 450 nm was measured by a microplate reader.
Cell adhesion assays were performed in a 24-well suspension culture plate (Iwaki). Purified ERE-D20 and ERE proteins (1000 nM) were added to culture plates and incubated for 2 h at 37°C. Then, the plate was washed with PBS three times. C3H10T1/2 cells suspended in FibroLife Serum-Free Medium (Lifeline Cell Technology) were added to the wells (8000 cells/well) and cultured for 6 h. Attached cells were evaluated with a Cell Counting Kit-8 (Dojindo).
The wells of a 96-well suspension culture plate (Sumilon, MS-8096R) were coated with ERE and ERE-D20 (100 or 1000 nM), respectively. After incubation for 2 h at 37°C with shaking, wells were washed with PBS-T followed by blocking with Blocking One overnight at 4°C. After washing with PBS-T, various concentrations of bFGF were added and plates were incubated for 1 h at 37°C. After washing with PBS-T, a solution of 1/1000 diluted rabbit anti-bFGF antibody (Sigma-Aldrich) was added and incubated for 1 h at 37°C. After washing with PBS-T, anti-rabbit IgG-HRP (Jackson ImmunoResearch Inc.) was added and reacted for 1 h at 37°C. After washing with PBS-T, TMB peroxidase substrate was added to the plate. Finally, 1 M HCl was added to stop the reaction and the absorbance at 450 nm was measured by a microplate reader.
The wells of a 24-well suspension culture plate (Iwaki) were coated with ERE-D20 (1000 nM) and incubated for 2 h at 37°C. After washing with PBS three times, bFGF (100 nM) was added to the wells and the plate was incubated for 2 h at 37°C. After washing with PBS, C3H10T1/2 cells suspended in FibroLife Serum-Free Medium without recombinant bFGF were added to wells (8000 cells/well) and cultured. During culture, media were changed every 2 days. The number of cells on days 1 and 5 was evaluated using a Cell Counting Kit-8.
Values are given as mean value ± standard deviation. Statistical analysis was performed by independent two-sample
The designed extracellular matrix, ERE, was genetically fused with 5 repeats of 4 aspartic acids and serine for tethering bFGF via electrostatic interaction (ERE-D20). To express ERE and ERE-D20 proteins,
SDS-PAGE analysis of purified ERE and ERE-D20 proteins. Lane M, SDS Broad Range Marker; lane 1, ERE protein; and lane 2, ERE-D20 protein.
First, adsorption of ERE-D20 onto the hydrophobic surface of the dish was evaluated. After addition of proteins to 96-well suspension culture plates, adsorbed proteins were detected with an anti-poly-histidine antibody. As shown in Figure
Adsorption of ERE-D20 on the hydrophobic surface of plates. Various concentrations of ERE protein (square) and ERE-D20 (circle) were coated on the surface of suspension culture plates, respectively. Error bars show the standard deviations of three independent measurements.
The ERE protein has a cell adhesion peptide, RGD, and it has shown cell adhesion activity. However, ERE-D20 has a negatively charged domain. Generally, cell surfaces are charged negatively. Therefore, the effects of D20 on cell adhesion activity were evaluated. Cells were seeded onto wells coated with ERE or ERE-D20 protein and incubated for 6 h. As shown in Figure
Cell adhesive activity of ERE-D20. (a) Evaluation of cell numbers after incubation for 6 h. Error bars show the standard deviations of three independent measurements. Statistically significant differences are indicated for
The interaction between ERE-D20 and bFGF was determined by ELISA using anti-bFGF antibody. Recombinant bFGF was added to wells of 96-well suspension culture plates coated with 100 nM ERE or ERE-D20. The tethered bFGF was detected using anti-bFGF antibody. As shown in Figure
Evaluation of bFGF tethered to ERE-D20. (a) Specific binding between basic domain of bFGF and negatively charged D20 of ERE-D20 (100 nM). (b) Concentration dependency of bFGF tethered to ERE-D20 (1000 nM). Error bars show the standard deviations of three independent measurements. Statistically significant difference is indicated for
Proliferation of cells cultured on bFGF-tethered ERE-D20 was evaluated. Cells were seeded into the wells of 24-well suspension culture plates coated with ERE-D20 alone and/or ERE-D20 tethered to bFGF. As shown in Figure
Induction of cell proliferation activities by culture on bFGF-tethered ERE-D20. Cells were cultured on ERE-D20 with or without tethered bFGF for 1 day (white bars) and 5 days (gray bars). Error bars show the standard deviations of three independent measurements. Statistically significant differences are indicated for
In our previous studies, growth factors were noncovalently immobilized on designed ECMs via helical peptides forming coiled-coil helical interaction [
In this study, we showed that bFGF could be tethered to our designed extracellular matrix (ERE-D20) via electrostatic interaction between the basic domain of bFGF and the acidic domain of ERE-D20. Cells cultured on bFGF-tethered ERE-D20 were well attached on ERE-D20 and underwent proliferation without addition of soluble bFGF. From these data, bFGF tethering to an ECM via electrostatic interaction could be applied for delivery of growth factors in tissue engineering.
The authors declare that there is no conflict of interests regarding the publication of this paper.