Human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex (East Rutherford, NJ). The cells were grown on endothelial cell medium EGM-2 (Cambrex) supplemented with 2 mM L-glutamine, 1000 U/mL penicillin, and 100 mg/L streptomycin (Sigma, St. Louis, MO), and they were used through passage 8. 10T1/2 cells (American Type Culture Collection, Rockville, MD) were grown and maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1000 U/mL penicillin, and 100 mg/L streptomycin, and they were used through passage 18. All cells were incubated at 37 °C in a 5% CO2 environment.
Synthesis of PEG-polymer hydrogel precursors
A MMP-sensitive peptide sequence, GGGPQG↓IWGQGK was synthesized by solid phase peptide synthesis based on standard Fmoc chemistry using an Apex396 peptide synthesizer (Aapptec, Louisville, KY, USA). Hydrogels with this peptide in the polymer backbone have been shown to be completely degraded by matrix metalloproteinases (MMP) [11
]. Following purification, synthesis of the peptide was confirmed with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF; Bruker Daltonics, Billerica, MA, USA)
Synthesis of the ABA block copolymers with the peptide linker was completed as described previously [10
]. Briefly, the MMP-sensitive peptide sequence was reacted with acrylate-PEG-N
-hydroxysuccinimide (acrylate-PEG-NHS, 3400 Da; Nektar) in a 1:2 (peptide: PEG) molar ratio in 50 mM sodium bicarbonate buffer (pH 8.5) for 2 h. This step conjugates a PEG-monoacrylate chain to the N-terminus and to the amine group on the lysine at the C-terminus of the peptide. The resulting product was then dialyzed (MWCO 5,000; Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) to remove unreacted peptide and PEG moieties, and the product was lyophilized, frozen, and stored under argon at - 80 °C. The cell adhesive ligand, Arg-Gly-Asp-Ser, (RGDS, American Peptide, Sunnyvale, CA) was conjugated to acrylate-PEG-NHS in 1:1 molar ratio under the similar conditions. Fluorophore-tagged PEG-RGDS was synthesized by reacting PEG-RGDS with Alexa Fluor-488 carboxylic acid (Invitrogen) in 10-fold dye molar excess in DMF. VEGF165
(Sigma) was conjugated to acrylate-PEG-SMC in 200:1 molar ratio at pH 8.5 at 4 °C for 4 days, followed by final 4 hours at 24 °C. The resulting PEG-VEGF solution was then lyophilized and reconstituted in HEPES buffered saline (HBS) with 0.1% BSA at 4°C until use. A gel permeation chromatography system equipped with UV-vis and evaporative light scattering detectors (Polymer Laboratories, Amherst, MA) was used to analyze the products.
Fabrication of MMP-sensitive PEG hydrogels
The hydrogel precursor solution was prepared in 10 mM HBS (pH 7.4) with PEG-RGDS (3.5 μmol/mL), a photoinitiator, Irgacure-2969 (0.3 mg/ml; Ciba Corporations, Basel, Switzerland), and varying amount of MMP-sensitive PEG-GGGPQGIWGQGK-PEG. In this work, MMP-sensitive PEG precursor was added to the final concentrations of 7.5 mg/ml, 10 mg/ml, 12.5 mg/ml, and 15 mg/ml to fabricate hydrogels with polymer weight percentages of 7.5%, 10%, 12.5%, and 15%, respectively. For HUVECs only culture, 6×106 cells were added to the hydrogel precursor solution. For HUVEC and 10T1/2 co-culture, 4.8×106 HUVECs and 1.2×106 10T1/2 cells were added. On coverglasses that had been cleaned with 70% ethanol and sterilized under UV lamp over night, the PEG precursor-cell suspension was dispensed as 5 μl droplets and photopolymerized by exposure to long-wavelength UV light (365 nm, 10 mW/cm2) for 7 min. The hydrogels were then immersed with EGM-2 media and incubated at 37 °C in a humidified atmosphere containing 5% CO2. For PEG hydrogels used in corneal angiogenesis assay in vivo, VEGF was mixed into the polymer solution to create concentrations of 512 ng VEGF per gel and 1.9 ng PEG-VEGF per gel.
Characterization of hydrogels
The swelling ratio and water content of the hydrogels were then determined as described previously [17
]. The compressive moduli of the different hydrogels with and without cells were determined using a 10 N load cell at a crosshead speed of 0.5 mm/min in an Instron-3342 (Canton, MA) mechanical tester as previously described [18
]. Degradation rates of MMP-sensitive PEG hydrogels formulated with varying polymer weight percentages were measured by monitoring the release of tryptophan (W) in the MMP-sensitive peptide sequence with a UV-Vis spectrophotometer (Carey 5000 Varian, Walnut Creek, CA) at 280 nm.
Measurement of tubule length and the number of branching points in 3D network of hydrogels
Endothelial tubule formation was evaluated by measuring total tubule length formed and the number of branching points over 6 days. On day 1, 3, and 6, cells in the hydrogels were stained with 2 μM calcein AM and visualized with a confocal microscopy (Zeiss LIVE 5, Carl Zeiss, Thornwood, NY) with z stack depth ranging 100-200 m. Scion Image (Scion Corporation, Frederick, MD) was used to trace and measure the total tubule length and the number of branching points in each area. The data were normalized to the z stack depth in each area to account for the different optical slice volumes of the images.
Time-lapse video confocal microscopy
To record time-lapse images during endothelial tubule formation, HUVECs and 10T1/2 were pre-labeled with 50 μg CellTracker Green CMFDA (5-chloromethylfluorescein diacetate; Invitrogen) and 50 μg CellTracker Red CMTPX (Invitrogen), respectively, per manufacturer's instructions prior to encapsulation in the hydrogels. After photoencapsulation of the cells, the hydrogels were incubated in EGM-2 media for 5 hrs before time-lapse imaging. A confocal microscope (Zeiss LIVE 5) equipped with a motorized XYZ stage and a temperature hood was used to record time-lapse images. T he temperature hood was perfused with 5% CO2 during the experiments. Confocal images were obtained at 1 hr intervals for 66 hrs, and the motorized stage allowed automatic collection of images from pre-recorded, multiple locations.
Isolation of cellular lysates and Western blotting
The hydrogels were prepared as 5 μl droplets each with either HUVEC monoculture (24×106 cells/ml), 10T1/2 mono-culture (6×106 cells/ml), or HUVECs and 10T1/2 co-culture (24×106 and 6×106 cells/ml, respectively). After 6 days, the cells were isolated from the bulk of hydrogels by degrading away the hydrogels with 2 mg/ml collagenase-1 solution (Sigma) for 10 min at 37 °C. Cell suspensions from four 5 μl hydrogel droplets were pooled for each cell culture condition to obtain enough cell lysate for Western blotting. In parallel experiments, cells were plated on 6 well tissue culture plates with either HUVEC mono-culture (24×104 cells/well), 10T1/2 mono-culture (6×104 cells/well), or HUVECs and 10T1/2 co-culture (24×104 and 6×104 cells/well, respectively). SDS-PAGE and Western blotting were performed following a stand protocol. The primary antibodies used for Western blotting include antibodies against smooth muscle α-actin (SM-αactin, 1:3000; ABCam, Cambridge, MA), calponin (1:500; ABCam), and caldesmon (1:500; Sigma). The secondary antibody was used at 1:2000, 1:1000, and 1:1000 dilutions, respectively.
Immunofluorescence staining was performed to confirm differentiation of 10T1/2 cells into SMC lineages and to visualize deposition of ECM proteins adjacent to the tubular structures. On day 1 and 6, the hydrogels containing HUVEC and 10T1/2 co-culture were fixed, permeabilized, and stained with primary and secondary antibodies. The primary antibodies used were mouse anti-SMα-actin (Sigma), rabbit anti-CD31 (Bethyl), rabbit anti-collagen-type IV (ABCam), and rabbit anti-laminin (Sigma) IgGs. The secondary antibodies were anti-rabbit and anti-mouse antibodies conjugated with either Alexa flour 488, Alexa fluor 594, or rhodamine (Invitrogen). For some samples, actin cytoskeletons and nuclei were stained with TRITC-conjugated phalloidin (5 U/ml, Sigma) for 1 h and DAPI (300 nM, Invitrogen) for 5 min.
Hydrogel implantation into the mouse cornea
Using a modified version of the previously described corneal micropocket angiogenesis assay [19
], hydrogels were implanted into mouse cornea in Flk1-myrmCherry
transgenic mouse, which exhibits EC-specific expression of a myristoylated mCherry fluorescence protein in EC membrane [20
]. Briefly, mice were anesthetized, and a partial thickness incision was made into the mouse cornea. The micropocket was created using a von Graef knife, and hydrogels were implanted into the micropocket immediately after UV photopolymerization. Seven days post-implantation, some mice were injected intravenously with dextran-Texas red (MW 70KDa), and the mice were sacrificed with CO2
asphyxiation. Eyes from the mice were enucleated and fixed in 4% paraformaldehyde for one hour at 4°C. Corneal flat-mount preparations were made, and imaging was performed using a Zeiss LSM 510 META inverted microscope system (Carl Zeiss Inc) equipped with a Zeiss Plan-Apochromat 20×/0.75 NA objective. 543-nm and 488-nm lasers were used to excite the Flk1-myrmCherry
and fluorophore-tagged PEG-RGDS, respectively.
Statistical analysis was performed with Jmp 5.1 (SAS Institute Inc, Cary, NC). Data sets were analyzed using two-way analysis of variance (ANOVA), followed by Tukey's HSD test for multiple comparisons. P-values less than 0.05 were considered statistically significant. All values are reported as mean ± standard deviation.