2.1. Synthesis of methacrylated heparin
The methacrylated heparin was prepared by reacting heparin (Mw 17,000, Sigma, St. Louis, MO) with 2-aminoethyl methacrylate (AEMA, Sigma). To synthesize the methacrylated heparin with theoretical methacrylation of two carboxylic acid groups, heparin (1 g) was dissolved in a buffer solution (1% w/v, pH 6.5) of 50 mM 2-morpholinoethanesulfonic acid (MES, Sigma) containing 0.5 M NaCl. N-hydroxysuccinimide (NHS, 13.8 mg; Sigma) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC, 45.1 mg; Sigma) (molar ratio of NHS:EDC=1:2) were added to the solution to activate the carboxylic acid groups of the heparin. After 5 min, AEMA (21.7 mg) (molar ratio of NHS:EDC:AEMA=1:2:1) was added to the product and the reaction was maintained at room temperature for 24 h. The mixture was precipitated with the addition of excess acetone, dried under reduced pressure, and rehydrated to a 1% w/v solution in ultrapure deionized water (diH2O) for further purification. The methacrylated heparin was purified by dialysis (MWCO 3500; Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) against diH2O for 3 days, filtered (0.22 μm filter), and lyophilized. To verify the methacrylation of heparin, unmodified and methacrylated heparin were dissolved in deuterium oxide (D2O, Sigma) and placed in separate NMR tubes. The 1H-NMR spectra of the samples were recorded on a Varian Unity-300 (300 MHz) NMR spectrometer (Varian Inc., Palo Alto, CA, USA) using 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (Sigma) as an internal standard.
2.2. Photocrosslinking for physical characterization of hydrogels
Low molecular weight sodium alginate (37,000 g/mol) was prepared by irradiating Protanal LF 20/40 (196,000 g/mol, FMC Biopolymer, Philadelphia, PA, USA) at a gamma dose of 5 Mrad. Methacrylated alginate (ALG) [13
] and RGD-modified methacrylated alginate (RGD-ALG) at a theoretical methacrylation of 45% (25% actual) were prepared as previously reported [16
]. To fabricate photocrosslinked HP-ALG hydrogels, methacrylated alginate (0.182 g) and methacrylated heparin (0.018 g) were dissolved in 10 ml of diH2
O or Dulbecco’s Modified Eagle Medium (DMEM, Sigma) with 0.05% w/v photoinitiator (Irgacure-2959, Sigma). These solutions were placed between two glass plates separated by 0.75 mm spacers and photocrosslinked with 365 nm UV light (Model ENF-260C, Spectroline, Westbury, NY) at ~1 mW/cm2
for 10 min to form the hydrogels. To fabricate ALG hydrogels without heparin as a comparative group, methacrylated alginate (0.2 g) was dissolved in 10 ml of diH2
O or DMEM with 0.05% w/v photoinitiator. These solutions were photocrosslinked as described above. Photocrosslinked hydrogel disks were created using a 6 mm diameter biopsy punch and placed in diH2
O or DMEM for mechanical testing, swelling, and degradation studies. To verify the completeness of methacrylated heparin and methacrylated alginate photocrosslinking, methacrylated alginate (0.0182 g) and methacrylated heparin (0.0018 g) were dissolved in D2
O (Sigma) with 0.05% w/v photoinitiator, placed in an NMR tube, and photocrosslinked as described above. The 1
H-NMR spectra of the HP-ALG before and after crosslinking were then determined as described above.
2.3. Mechanical testing
The elastic moduli of the photocrosslinked HP-ALG or ALG hydrogels formed with diH2O or DMEM were determined by performing constant strain rate compression tests using a Rheometrics Solid Analyzer (RSAII, Rheometrics Inc., Piscataway, NJ, USA) equipped with a 10 N load cell. The photocrosslinked HP-ALG or ALG hydrogel disks were prepared as described above and maintained in DMEM or diH2O at 37 °C. After 24 h incubation in DMEM or diH2O, swollen hydrogel disks were punched once again to form 6 mm diameter disks, their thickness was measured using calipers, and uniaxial, unconfined compression tests were performed on the hydrogel disks at room temperature using a constant strain rate of 5%/s. Elastic moduli of photocrosslinked HP-ALG hydrogels were determined from the slope of stress vs. strain plots, limited to the linear first 5% strain of the plots (N=3 for each condition).
2.4. Swelling and in vitro degradation of HP-ALG hydrogels
The photocrosslinked HP-ALG or ALG hydrogels were lyophilized and initial dry weights (Wi) were measured. Dried hydrogel samples originally formed with diH2O or DMEM were immersed in 50 ml of diH2O or DMEM, respectively, and incubated at 37 °C to reach equilibrium swelling state. The diH2O or DMEM was replaced every week. Over the course of 8 weeks, samples were removed, rinsed with diH2O, and the swollen (Ws) hydrogel sample weights were measured. The swelling ratio (Q) was calculated by Q=Ws/Wi (N=3 for each condition per time point). After weighing the swollen hydrogels, they were lyophilized and weighed (Wd). The percent mass loss was calculated by (Wi−Wd)/Wi ×100 (N=3 for each condition per time point).
2.5. Release kinetics of growth factors
The release kinetics of four different growth factors [FGF-2 (generously provided by the NCI BRB Preclinical Repository), VEGF (generously provided by the NCI BRB Preclinical Repository), TGF-β1 (PeproTech Inc., Rocky Hill, NJ), and BMP-2 (GenScript, Piscataway, NJ)] from HP-ALG, ALG, HP-RGD-ALG, and RGD-ALG hydrogels were determined. ALG (27.3 mg) and methacrylated heparin (2.7 mg) or RGD-ALG (27.3 mg) and methacrylated heparin (2.7 mg) were dissolved in diH2O (1.5 ml) with 0.05% w/v photoinitiator. One of four different growth factors (0.75 μg) was added to the alginate solutions. After gently mixing for 5 min, aliquots (300 μl) of solution were placed in 96-well tissue culture plates and photocrosslinked with 365 nm UV light at ~1 mW/cm2 for 10 min to form the hydrogels. To prepare the ALG or RGD-ALG hydrogels, ALG (30 mg) or RGD-ALG (30 mg) was dissolved in diH2O (1.5 ml) with 0.05% w/v photoinitiator. Each of four different growth factors (0.75 μg) and unmodified heparin (18.75 μg) were added to the methacrylated alginate solution. After gently mixing for 5 min, aliquots (300 μl) of solution were photocrosslinked as described above. Each photocrosslinked hydrogel was then placed in a 15-ml conical tube containing 10 ml phosphate buffered saline (PBS, pH 7.4) and incubated at 37 °C. At predetermined time points over the course of 3 weeks, the supernatant was withdrawn and fresh buffer was replenished. The amount of growth factor in the supernatants was determined with sandwich enzyme-linked immunosorption assay (ELISA) kits (FGF-2, VEGF, and TGF-β1: Duoset, R&D Systems; BMP-2: Human BMP-2 Construction kit, Antigenix America Inc., New York, NY, USA). ELISA plates were coated with capture antibodies according to the manufacturer’s instructions, and were blocked with bovine serum albumin (1 % w/v) and sucrose (5 % w/v) for 1 h. After the appropriately diluted samples were added to the ELISA plates, bound growth factors were detected using biotinylated anti-human antibodies. Then, streptavidin-conjugated horseradish peroxidase was added to the plates. The substrate (tetramethylbenzidine) was subsequently added and incubated for 20 min. The enzyme reaction was stopped by addition of an acidic solution (2 N H2SO4). The absorbance of the samples was read at 450 nm on a plate reader (SAFIRE, Tecan, Austria). The amount of growth factor present in each sample was determined using calibration curves derived using known concentrations of the growth factors (N=3 for each condition per time point).
2.6. Bioactivity assessment of released growth factors in vitro
The bioactivity of VEGF released from HP-ALG hydrogels in vitro was examined by determining its ability to stimulate the proliferation of human umbilical vein endothelial cells (HUVECs, passage number 3; a generous gift from Andrew Putnam, Ph.D.) cultured in endothelial cell basal medium-2 (EBM-2, Cambrex Bio Science Inc., Walkersville, MD, USA) with 2% fetal bovine serum (FBS, Cambrex Bio Science Inc.) in a humidified incubator at 37 °C with 5% CO2. 1 μg of VEGF was loaded into the HP-ALG hydrogels, prepared as in Section 2.5. Cells (1×105 per well) were plated in each well of six-well tissue culture plates in 3 ml EBM-2+2% FBS, and 1 h hour later the VEGF-loaded hydrogels were placed on culture inserts (Transwell®, Corning Incorporated) in each well. The medium was changed every 3 days. At 1, 2, and 3 weeks, cell numbers were measured using a hemacytometer. ALG hydrogels containing 1 μg of VEGF and 25 μg of unmodified heparin in 3 ml EBM-2 with 2% FBS served as a comparative group. An additional comparative group without hydrogels was composed of VEGF (300 ng) with unmodified heparin (7.5 μg) supplied to HUVEC cultures in 3 ml EBM-2 with 2% FBS; these cultures were therefore exposed to 2.1 μg soluble VEGF over the course of 3 weeks. HUVECs cultured without hydrogels in endothelial cell growth medium (EGM-2, Cambrex Bio Science Inc.) or EBM-2 without VEGF served as positive and negative controls, respectively (N=3 for each condition per time point).
The bioactivity of BMP-2 released from HP-ALG hydrogels in vitro was assessed by determining its ability to stimulate the alkaline phosphatase (ALP) activity of MC3T3-E1 Subclone 4 cells (ATCC CRL 2593, Manassas, VA, USA), a mouse preosteoblast cell line, cultured in DMEM containing 10% FBS. 1 μg of BMP-2 was loaded into each photocrosslinked HP-ALG hydrogel, which was formed as in Section 2.5. Cells (3×104) were plated in each well of six-well tissue culture plates with 3 ml DMEM+10% FBS, and 1 h later the BMP-2-loaded hydrogels were placed on culture inserts in each well. The medium was changed every 3 days. BMP-2 (300 ng) mixed with 25 μg unmodified heparin added to MC3T3 cell cultures in 3 ml DMEM with 10% FBS served as a positive control. MC3T3 cells cultured in DMEM with 10% FBS and HP-ALG hydrogels without BMP-2, and DMEM with 10% FBS without either HP-ALG or BMP-2 served as a comparative and a negative control group, respectively (N=3 for each condition per time point). At predetermined time points over 2 weeks, the hydrogels were removed, and the ALP activity of the cells was measured using SensoLyte® pNPP ALP Assay kit (AnaSpec Inc., Fremont, CA) according to manufacturer’s instructions. At each time point, cells were lysed by adding 1 ml lysis buffer solution, and the lysates were cleared by centrifugation for 10 min at 16,200×g using an ultracentrifuge. 50 μl of supernatant was incubated with 50 μl of ALP substrate containing p-nitrophenylphosphate (pNPP) at room temperature for 30 min. The reaction was stopped by adding 50 μl of stop solution to the substrate reaction solution. The absorbance of the samples was read at 405 nm on a plate reader. Each ALP activity measurement was normalized to total sample protein content, which was quantified using a BCA protein assay kit as per the manufacturer’s instructions (Pierce Chemical, Rockford, IL, USA) (N=3 for each condition per time point).
2.7. In vivo bone formation
The capacity of the system to promote in vivo bone formation by host cells was tested. The following conditions were examined: BMP-2 (1 μg/hydrogel disk) loaded in HP-ALG hydrogels, BMP-2 (1 μg/hydrogel disk) mixed with unmodified heparin (25 μg/hydrogel disk) loaded in ALG hydrogels, and HP-ALG hydrogels without BMP-2. SCID mice (ICRSC, 4–5 weeks old; Taconic, city, USA) were anesthetized with xylazine (20 mg/kg) and ketamine (100 mg/kg), 2 small skin incisions were made on the right and left sides of the backs of the mice to create subcutaneous pouches, and photocrosslinked hydrogel disks (diameter=6 mm) were immediately implanted into the pouches (2 implants per mouse, N=10 implants per condition). Subsequently, the skin was closed with 6–0 silk sutures (Ethicon, Lenneke Marelaan, Belgium). Eight weeks after implantation, the mice were sacrificed, and the implants were retrieved. Three implants per condition were used for histomorphometric analysis and another three were used for quantification of total calcium content. The histological specimens were fixed in formalin, embedded in paraffin, sectioned at a thickness of 4 μm, and examined with hematoxylin and eosin (H&E) staining and Goldner’s trichrome staining. The bone formation (%) was calculated using three different Goldner’s trichrome stained images according to the following equation: Bone formation (%)=presence of bone on the perimeter of implant (mm)/perimeter of implant (mm)×100. The amount of calcium deposited in and directly surrounding the implants was measured using a calcium assay kit (Calcium Reagent Set, Pointe Scientific Inc., Canton, MI, USA) according to the manufacturer’s instructions. All animal procedures were carried out in accordance with a protocol approved by the Institutional Animal Care and Usage Committee at Case Western Reserve University.
2.8. Statistical analysis
All quantitative data are expressed as the mean±standard deviation. Statistical analysis was performed with one-way analysis of variance (ANOVA) with Tukey honestly significant difference post hoc test using Origin software (Origin Lab Co., Northampton, MA). A value of p<0.05 was considered statistically significant.