2.1. Synthesis of PEG based macromers
All chemicals were purchased from Sigma-Aldrich (Milwaukee, WI) unless otherwise noted. PEG based macromers were synthesized by the reaction represented in . Briefly, PEG with nominal molecular weight of 200 Da was dried by azeotropic distillation in toluene and remaining toluene and traces of water were further removed under reduced pressure. Sebacoyl chloride was purified by distillation at boiling point (168 °C) under vacuum (6mm Hg). A total of 100 g of dried PEG was dissolved in 500 ml of anhydrous methylene chloride. Then 50% molar excess of triethylamine was added dropwise into the solution with vigorous stirring followed by dropwise addition of sebacic chloride (0.9 mole/mole of PEG) solution in an ice bath. The reaction mixture was stirred for 24 h at room temperature. The solvent was removed by rotovaporation and the product was dissolved in warm ethyl acetate. Triethylamine hydrochloride salt was removed by filtration until the solution was clear and the final product (namely PEGS) was precipitated in petroleum ether and dried under vacuum.
Synthesis and photopolymerization of PEG based macromers incorporated with dicarboxylic acids and terminated with acrylate group.
To prepare PEG sebacate diacrylate (PEGSDA), 50 g of PEGS was dissolved in methylene chloride. Again 50% molar excess triethylamine was added dropwise into the solution, followed by dropwise addition of dried acryloyl chloride (50% molar excess of PEGS). The reaction was run for another 24 h at room temperature and the product was dissolved in ethyl acetate, filtered, and precipitated in petroleum ether. To obtain the final product, the macromer was dissolved in methylene chloride, reprecipitated in dry petroleum ether twice, and dried under vacuum overnight.
PEG terephthalate (PEGT) was similarly synthesized by using PEG (MW: 200) and terephthaloyl chloride and PEG terephthalate diacrylate (PEGTDA) was obtained by end capping PEGT with acrylate group as described above.
2.2. Macromer characterization
Molecular weight of the synthesized polymers were measured by gel permeation chromatography (GPC, Waters, Milford, MA) equipped with a HPLC pump and a refractive index detector by injecting 20 μl of the sample and eluting at 1 ml/min of flow rate in tetrahydrofuran (THF) (Fisher, Pittsburgh, PA). Monodisperse polystyrene standards (Polyscience, Warrington, PA) with number average molecular weight (Mn) of 0.474, 6.69, 18.6, and 38 kDa and polydispersity index (PDI) of less than 1.1 were used to construct the calibration curve. Mn and weight average molecular weight (Mw) were calculated from the standard curve.
The characterization of hydroxyl terminated polymers (PEGS and PEGT) and acrylate terminated polymers (PEGSDA and PEGTDA) were performed by using Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR, Nicolet 8700, Madison, WI) and proton nuclear magnetic resonance (1H-NMR, Bruker AC 500, Billerca, MA) spectroscopy. For FTIR measurement of crosslinked PEG based networks, all samples were soaked in methylene chloride for 24 h at room temperature to completely remove all possible contaminants or unreacted monomers and then the spectra were obtained.
Thermal properties of the macromers were characterized using a differential scanning calorimeter (DSC, TA instruments, New Castle, DE). To keep the same thermal history, the samples were first preheated from room temperature to 100 °C at a rate of 10 °C/min in a nitrogen atmosphere and then cooled at a rate of 5 °C/min to −90 °C and the scan was then recorded from −90 °C to 100 °C.
The photoinitiator, Igracure 2959 (Ciba Specialty Chemicals, Tarrytown, NY), was added to the polymer at a final concentration of 0.05% (w/v). The mixture was then incubated in the convection oven at 60 °C for 10 min to remove air bubbles. The solution was pipetted into glass plates with a 0.8 mm spacer and exposed to UV light (λ = 310 – 400 nm, Blak-Ray®, Upland, CA) for 10 min. The crosslinked polymers were then removed from the glass plate and cut into disks (0.8 cm × 0.8 mm) with a cork borer.
To incorporate HEMA into the polymer network, up to 30 % (w/w) of HEMA was added into the melted polymer (PEGSDA or PEGTDA) then the mixture was photopolymerized as described above. To calculate the conversion of crosslinked network, initial solution was weighed as Wbc and the final weight of the crosslinked network was weighed as Wac and the percent conversion was then calculated as Wac/Wbc × 100. The sol fraction was also determined to measure the fraction of unreacted macromer. The crosslinked discs were weighed as Wis and the dry discs were soaked in methylene chloride for 24 h at room temperature to completely dissolve the unreacted macromers and weighed as Wfs. The percent sol fraction was then calculated as (Wis − Wfs)/Wis × 100.
2.4. Swelling and degradation
After photopolymerization, the samples were incubated in pH 7.4 phosphate buffered saline (PBS) at 37 °C with gentle shaking. Swollen samples were weighted as Ws at different time points and dry weight (Wd) was measured after lyophilization under vacuum for 24 h. The swelling ratio was then calculated as (Ws − Wd)/Wd. In separate experiments, the swollen samples were lyophilized and weighed (Wi) and the dried samples were incubated at 37 °C in PBS or in 1.0 N NaOH solution for accelerated degradation on an orbital shaker. At certain time points, specimens were rinsed with deionized water, lyophilized and weighed (Wd2). The percent weight loss after lyophilization was calculated as (Wi − Wd2)/Wi × 100.
2.5. Mechanical properties
Mechanical properties of the crosslinked polymers were analyzed with a dynamic mechanical rheometer (TA Instruments, New Castle, DE) using the parallel-plate setup and a dynamic mechanical analyzer (TA instruments). All samples were tested at room temperature after 24 h of equilibration in deionized water. Dynamic shear strain sweep experiment was first performed at a frequency of 10 rad/s to determine the linear stress-strain range of the different samples. The results indicated that 0.1% of shear strain was in the linear stress-strain range of tested samples. The dynamic shear elastic modulus of the samples was then obtained by the frequency sweep test with a frequency range from 1 to 100 rad/s at a strain of 0.1%.
The crosslinking density of these macromers was then calculated by the following equation (1) 16,23
Where n represents the crosslink density which is moles of active network chains per unit volume, Mc is the molecular weight between crosslinks, R is the universal gas constant (8.3144 J/mol·K), T is the absolute temperature (K), ρ is the macromer density (g/m3) and G is the shear modulus.
Static elastic compressive modulus was measured for cylindrical samples by a dynamic mechanical analyzer (TA instruments). Before testing, initial cross-section area and dimension of each sample were measured. All samples were submerged in de-ionized water and compressed at a loading rate of 2 N/min up to 15 N. Compressive modulus was determined by dividing applied forces and dimensional changes into initial cross-section area and dimension, and then calculating the slope of linear region in the stress-strain curve.
Rat bone marrow stromal cells (MSCs) were obtained as previously described 24
. Briefly, MSCs were isolated from femurs of five month old male Sprigue-Dawley rats (Harlan, Indianapolis, IN). The femora were harvested, soft tissue was removed, and the femora were placed in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (50 μg/ml), streptomycin (50 μg/ml), neomycin (100 μg/ml), and fungizone (25 μg/ml). The femora were rinsed with sterile PBS, and the epiphyses were cut off. The bone marrow was flushed out using a syringe with an 18 gauge needle inserted into the diaphysis. The marrow was flushed with medium into a sterile 50 ml centrifuge tube, titrated with the syringe to form a single cell suspension, and plated in two T-75 flasks. The cells were then cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO2
atmosphere. Cells were used between passages three to six. Additionally, PC-12 cells were routinely cultured in DMEM supplemented with 10% FBS and 10% horse serum at 37 °C in a 5% CO2
The cells (MSCs or PC-12 cells) were seeded at a density of 20,000 cells/cm2 on the bottom of the 24-well transwell tissue culture plates (Costar, membrane pore size: 0.3 μm, Pittsburgh, PA) and incubated for 24 h. PEGSDA and PEGSTA disks (5 mm diameter, 0.8 mm thick) were then placed into the inserts of the transwells and the cells were further cultured for up to 7 days in the presence of the polymers. The polymer disks were sterilized by incubation with 70% ethanol for 1 h followed by extensive washes with sterile PBS. Transwell inserts without the polymer disks were also transferred to the culture plates containing the cells and used as the positive control.
On days 4 and day 7, cell viability was evaluated using a colorimetric cell metabolic assay (CellTiter 96 Aqueous One Solution, Promega, Madison, WI), based on the MTS tetrazolium compound, 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. The reagent solution was added to each well and incubated for 1 h at 37 °C. The supernatant from each well was transferred to a 96 well plates. The UV absorbance at 490 nm was measured using a plate reader (SpectraMax Plus, Molecular Devices, Sunnyvale, CA), which is correlated to the viable cell number, and inversely correlated to the toxicity of the crosslinked polymer disks.
2.7. Statistical analysis
All data are represented as means ± standard deviations for n = 3. Statistical analysis was performed using single factor analysis of variance (ANOVA) with Scheffe’s posthoc test at a significance level of 0.05.