Isolation of Chondrocytes
Chondrocytes were isolated from articular cartilage of 2–4-week-old calf following the previously reported method (12). Briefly, chondrocytes were isolated by incubating cartilage pieces in high glucose Dulbecco’s modified Eagle medium (DMEM) (GIBCO) containing 5% fetal bovine serum (FBS) (Atlanta Biologicals, GA) and 0.2% collagenase (Worthington Biochemical Corp., NJ) for 16 h at 37°C and 5% CO2. The resultant cell suspension was filtered through a 70 μm nylon mesh, and the cells were washed with phosphate-buffered saline (PBS) containing 100 U/mL of penicillin and 100 μg/mL of streptomycin (pen-strep) (GIBCO).
Synthesis of PEGDA Precursors
PEGDA was synthesized by reacting acryloyl chloride with hydroxyl end groups of PEG using the reported method (19). Briefly, 3 mmol of PEG was transferred to a round-bottom flask containing 500 mL of toluene and refluxed in an oil bath for azeotropic removal of traces of water. On cooling this solution to room temperature, 12 mmol of triethyl amine was added with vigorous stirring. Twelve mmol of acryloyl chloride in 15 mL anhydrous tetrahydrofuran was added to this reactant mixture drop-wise for 30 min in ice-bath. The content was then heated at 45°C in oil bath overnight. Quaternary ammonium salt formed was removed by filtering the solution through a bed of diatomaceous earth. The filtrate was concentrated on a rotary evaporator and precipitated in diethyl ether. Precipitated product was re-dissolved in dichloromethane and re-precipitated in diethyl ether. Resultant PEGDA precursor was column-purified and dialyzed against deionized water followed by lyophilization before use. PEGDA with molecular weights of 3,400 Da (PEGDA-3.4 k), 6,000 Da (PEGDA-6 k), 10,000 Da (PEGDA-10 k), and 20,000 Da (PEGDA-20 k) were used for this study. Dialysis tubing (Spectrum Labs Inc., USA) with a molecular weight cut-off (MWCO) of 500 Da was used for PEGDA-3.4 k, -6 k and -10 k, while for PEGDA-20 k, a dialysis tube with a MWCO of 12,000 Da was used.
Photopolymerization and Chondrocyte Encapsulation
For photopolymerization, a 10% (w/v) solution of PEGDA in PBS was mixed with 0.05% (w/v) photoinitiator Irgacure 2959 (CIBA Speciality Chemicals, USA) used as 10% (w/v) solution in 70% ethanol. The polymer solution was transferred to cylindrical molds (having dimensions of 6.5 mm diameter and 4 mm height) and exposed to 365 nm UV radiation for 5 min. For cell encapsulation, primary chondrocytes were dispersed in sterile polymer solution to yield a final cell density of 20 × 106 cells/mL. Resulting cell-laden constructs were transferred to a 24-well plate and cultured in chondrocyte medium.
Swelling Ratio and Determination of Mesh Size
Swelling ratios of the acellular hydrogels were determined using gravimetric method. Hydrogels were thoroughly washed and swelled in PBS, then dried at 40°C under vacuum until a constant weight was obtained. Dried gels were re-immersed in PBS at 37°C, and the weight of swollen hydrogels was measured in an equilibrium-swollen state. Swelling ratio was calculated by using the following formula: \( {\hbox{SR = }}{{\hbox{W}}_{\rm{s}}}/{{\hbox{W}}_0} \), where W0 and Ws are weights of hydrogel before and after swelling, respectively. The average and standard deviation of triplicate samples was calculated.
The average molecular weight between the two adjacent crosslinks \( ({\overline M_c}) \) was determined using the Peppas-Merrill model following formfula given below (20):
$$ \frac{1}{{\overline {{M_c}} }} = \frac{2}{{\overline {{M_n}} }} - \frac{{\left( {\frac{{\bar{\upsilon }}}{{{V_1}}}} \right)\left[ {\ln (1 - {\upsilon_{2,s}}) + {\upsilon_{2,s}} + {\chi_1}\upsilon_{2,s}^2} \right]}}{{{\upsilon_{2,r}}\left[ {{{\left( {\frac{{{\upsilon_{2,s}}}}{{{\upsilon_{2,r}}}}} \right)}^{ \frac{1}{3} }} - \frac{1}{2}\left( {\frac{{{\upsilon_{2,s}}}}{{{\upsilon_{2,r}}}}} \right)} \right]}} $$
where \( {\overline M_n} \) is the average molecular weight of PEG oligomers, \( {\overline M_c} \) is the average molecular weight between two adjacent crosslinks, υ
2,s
is the polymer volume fraction in the swollen state, χ1 is the Flory-Huggin’s polymer–solvent interaction parameter (0.426 for PEG-water system), \( \overline v \) is the specific volume of PEGDA in its amorphous state (0.893 cm3/g), V1 is the molar volume of the solvent (18 cm3/mol for water), and υ
2,r
is the polymer fraction in the gel (0.1).
Mesh size (ξ) of the hydrogel is calculated by using the following formula (20):
$$ \xi = {(\overline {r_0^2} )^{1/2}}\upsilon_{2,s}^{ - 1/3} $$
$$ (\overline {{r_0}^2} ) = {l^2}\left[ {2\frac{{{{\overline M }_c}}}{{{M_r}}}} \right]{C_n} $$
where \( {[r_0^2]^{1/2}} \) is the root mean square end-to-end distance of the polymer in its free state, l is the carbon-carbon bond length (0.154 nm), Cn is the rigidity factor of polymer (4 for PEG), and Mr is the molecular weight of repeating units (44 g/mol for PEG).
Scanning Electron Microscopy (SEM)
The morphology and internal structure of the hydrogels were examined using scanning electron microscope (SEM, Philips XL30 ESEM). The equilibrium-swollen hydrogel samples were frozen in liquid nitrogen and immediately fractured. The fractured samples were lyophilized and finally sputter-coated with gold for 40 s by using Emitech K575X sputter coater.
Mechanical Properties
Mechanical properties of the hydrogels, such as compressive modulus, ultimate strength, and strain at break, were determined in their equilibrium-swollen state using the Instron 3342 Universal Testing system (Instron, Norwood, MA, USA) equipped with model 2519-004 force transducer. Cylindrical hydrogels with height and diameter of 7 mm and 8 mm, respectively, were used for testing. The maximum force load was 250 N, and the crosshead speed was 10 mm/min. Compressive modulus of the hydrogel was determined as the slope of the initial linear region of the stress–strain curve. Average and standard deviation of quadruplicate samples are reported.
Chondrocyte Culture
Chondrocyte medium was prepared by adding 10% FBS, 1 mM sodium pyruvate (GIBCO), 10 mM HEPES (GIBCO), 0.1 mM minimal essential medium with non-essential amino acids (GIBCO), 0.4 mM proline (Sigma), 50 mg/L vitamin C (Sigma), 100 U/ml of penicillin and 100 μg/mL of streptomycin to high glucose DMEM. The cell-laden hydrogels (hereafter referred to as constructs) were incubated at 37°C in humidified 5% CO2 atmosphere for four weeks. The medium was changed twice a week.
Live-Dead Assay
The cell viability of encapsulated chondrocytes was determined 24 h after encapsulation by using live/dead assay kit (Molecular Probes, Eugene, OR) following manufacturer’s protocol. Briefly, constructs sliced into thin sections were washed with PBS and incubated in a solution of 0.5 μL of Calcein AM and 2 μL of ethidium homodimer-1 in 1 mL DMEM. After an incubation of 30 min, the sections were rinsed with PBS, and the images were taken by using the Zeiss Observer A1 fluorescence microscope equipped with an X-Cite 120 (EXFO) mercury lamp.
Biochemical Assay
Constructs for biochemical assays were dried using a lyophilizer, and their dry weights were measured. Glycosaminoglycan (GAG), collagen, and deoxyribonucleic acid (DNA) contents were determined by using previously reported methods (21). Dried samples were digested with 1 mL papain solution (125 μg/mL) (Worthington Biochemical Corp., NJ) in PBE buffer at 60°C for 16 h.
DNA content of the papain digested samples was determined using Quant-iT PicoGreen dsDNA reagent (Molecular Probes). After incubation with the reagent, the fluorescence intensity was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. GAG content was measured using 1, 9-dimethylmethylene blue (DMMB) spectroscopic assay at 525 nm with chondroitin sulfate as a standard (22). For determination of collagen content, the papain digest was acid hydrolyzed and reacted with chloramine-T followed by p-dimethylaminobenzaldehyde (23). The collagen content was measured spectroscopically as hydroxyproline content at 550 nm. The conversion factor of 7.6 was used to convert the hydroxyproline to collagen content (24).
Histological and Immunofluorescent Assays
For histological and immunofluorescent assays, the constructs after four weeks of culture were fixed with 4% paraformaldehyde, embedded in optimum cutting temperature (OCT) compound (Ted Pella Inc, CA), and cryosectioned at 20 μm thickness. Safranin-O staining for proteoglycans was visualized using a microscope under bright field. Immunofluorescent staining for collagen type II was done by using polyclonal antibody 70R-CR008X (Fitzgerald Industries, MA) with final conjugation of bound antibody done by incubating with Alexa Fluor 488 goat anti-rabbit secondary antibody (Invitrogen, CA). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) solution (Vector Lab, CA).
Gene Expression by Qrt-PCR
Total RNA was isolated from the constructs using TRIzol and reverse transcribed into cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratory). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using power SYBR green PCR master mix solution and the ABI Prism 7300 Sequence Detection System (Perkin Elmer/Applied Biosystems, Rotkreuz, Switzerland). The primers used for qRT-PCR are listed in Table 1. The gene expression values of samples at four weeks were normalized to respective expression values of β-actin, and further normalized to day 0 values for comparison. Relative gene expression is then calculated as fold induction.
Table 1 qRT-PCR Primer Sequences
Statistical Analysis
Material properties of acellular PEGDA hydrogels were analyzed by one-way analysis of variance (ANOVA). For statistical comparison of biochemical assays between four groups, two-way ANOVA was done with Bonferroni post hoc test to identify significant differences between various constructs at respective time points. Both types of analysis were done at a ‘p’ value of 0.05.