Fabrication of a Double Crosslinked Interpenetrating. Polymeric Network (IPN) Hydrogel Surface Modified. with Polydopamine to Modulate Osteogenic

Transcription

1 Supporting information Fabrication of a Double Crosslinked Interpenetrating Polymeric Network (IPN) Hydrogel Surface Modified with Polydopamine to Modulate Osteogenic Differentiation of Adipose-Derived Stem Cells Settimio Pacelli, Kyle Rampetsreiter, Saman Modaresi, Siddharth Subham, Aparna R. Chakravarti, Stefan Lohfeld, Michael S. Detamore, Arghya Paul * Department of Chemical and Petroleum Engineering, BioIntel Research Laboratory, University of Kansas, Lawrence, KS, 66045, United States Biomechanics Research Centre (BMEC), Mechanical and Biomedical Engineering, College of Engineering and Informatics, National University of Ireland, Galway, H91 TK33 Ireland. Stephenson School of Biomedical Engineering, University of Oklahoma Norman, Oklahoma 73019, United States. * Corresponding author: Dr. Arghya Paul. arghyapaul@ku.edu S-1

2 Figure S1 1 H-NMR spectra of gelatin and GelMA displaying the successful introduction of methacrylic groups. The yellow arrow in the GelMA spectra indicates the protons of the double bond of the methacrylic groups, which are absent in the gelatin spectra. Figure S2. Viscosity measurements of alginate solutions prepared in water at 25 C. The viscosity of GelMA 6% and Alginate 1% w/v was selected as the optimal one to obtain homogeneous polymeric solutions prior UV irradiation. S-2

3 Figure S3. Representative frequency sweep profiles carried out at 25 C to study the effect of calcium chloride as physical crosslinker of GelMA alginate hydrogels. CaCl 2 was tested in the range of 0 up to 10 mm. Figure S4. Effect of calcium concentration on cell viability and morphology. A) Calcein staining of HUVECs seeded on IPN 6% containing different concentration of calcium chloride after 24 hours. B) Quantification of cell viability after 24 hours post seeding. A decrease in cell viability was evident for calcium concentration higher than 1 mm. Results are reported as mean ± standard deviation (n=5). * = p < S-3

4 Figure S5. Strain sweep profiles of the different IPN systems in the range of strain from 1 to 200% of strain at 1 Hz. The IPN 6% showed lower yield strain values with an increase in the slope of tan δ curve for lower strains values compared to the other systems. Figure S6. A) SEM images of cross-sections of IPN of GelMA 6% w/v and alginate 1% w/v hydrogels with and without a polydopamine (pda) coating. Scale bar 300 µm. B) Quantification of the pore size average for both groups by image J. Results are reported as the mean ± standard deviation (n=5). S-4

5 Figure S7. A) Schematic is representing the process of PCL scaffold coating with the IPN hydrogel. B) i) Image of the PCL scaffold uncoated and coated with the gel (on the right), ii) SEM image of the PCL scaffold uncoated and iii) coated with the hydrogel. Scale bar = 500 µm. iv) A picture displaying the construct used to fabricate the PCL scaffold. Table S1. Elementary composition of the surface in the different hydrogels obtained from the EDX spectra. Fluorine is only present in the Gel/Alg/Dop-Dex group. GelMA/Alg GelMA/Alg/Dop GelMA/Alg/Dop-Dex C 63.2% ± % ± % ± 3.8 N 19.5% ± % ± ± 3.1% O 17.4 ± 1.8% 20.7 % ± % ± 1.6% F % ± 0.8 S-5

6 Materials: Gelatin A from porcine skin (300 Bloom grade), methacrylic anhydride, anhydrous dopamine hydrochloride and Irgacure hydroxy-4 -(2-hydroxyethoxy)-2-methylpropiophenone were obtained from Sigma-Aldrich, (St. Louis, MO, USA). Alginate, Protanal LF 20/40 Mw: 196,000 Da was purchased from FMC BioPolymer (Philadelphia, PA). Tris-HCl, Molecular Biology Grade was purchased from Promega (Promega Corporation, Madison, WI, USA). Anhydrous calcium chloride was obtained from Acros Organics (NJ, USA). Dexamethasone was purchased from Tocris Bioscience (Bristol, UK). Dulbecco s Phosphate Buffered Saline (DPBS) by Gibco and Life Technologies and 2,4,6-trinitrobenzene sulfonic acid (TNBSA) was obtained from ThermoFisher (Rockford, IL, USA). Spectra/Por molecular porous membrane tubing (MWCO Kd) used for dialysis was purchased from Spectrum Laboratories, Incorporated (Rancho Dominguez, CA,USA). Synthesis and Characterization of Gelatin Methacrylamide (GelMA): Gelatin type A was chemically modified with methacrylic anhydride to introduce reactive methacrylic groups. Specifically, Gelatin 10% w/v was solubilized in DPBS ph 7.4 (100 ml) at 50 C for 1 hour. After solubilization, an excess of methacrylic anhydride (8 ml) was added dropwise to the polymer solution and allowed to react for 2 hours. Then, GelMA was diluted with DBPS (100 ml) and dialyzed using dialysis tubes (cut off 12,000-14,000 Da) against distilled water at 50 C. Water was changed twice a day for a week to ensure the complete purification of GelMA. After one week, GelMA was filtered using a filter with a pore size of 0.22 µm and frozen at -80 C before freezedrying. The successful methacrylation of gelatin was investigated through 1 H-NMR spectroscopy in D 2 O. Samples were prepared by solubilizing the polymer (10 mg) in 0.7 ml of D 2 O. Spectra were recorded with a Bruker Avance 400 MHz. Quantification of the degree of methacrylation was carried out using the TNBSA quantification assay [15]. Briefly, freeze-dried GelMA (0.2 mg/ml) was solubilized in 0.1 M sodium bicarbonate, (ph 8.5). TNBSA 0.01% w/v was added to the solution of GelMA, and the reaction was run at 37 C S-6

7 for 2 hours before addition of 1 N HCl. Absorbance was then read at 335 nm, and the percentage of unreacted amine groups was quantified based on the difference in the absorbance values resulting from the reaction of TNSBA 0.01% with gelatin at the same concentration of GelMA. IPN hydrogels fabrication: The hydrogels were prepared following a straightforward procedure. Briefly, alginate solution 2.5 % w/v was obtained by solubilizing the polymer alginate in distilled water at 60 C. Then, two different stock GelMA solutions at the concentrations of 6 % w/v and 12 % w/v were prepared by solubilizing the necessary amount of GelMa freeze-dried powder into distilled water at 50 C. The photoinitiator Irgacure 2959 solution 1% w/v was solubilized in PBS and stored in the dark. The three components were mixed in a volume ratio gelatin/alginate/photoinitiator (5:4:1) in order to obtain hydrogels with a final concentration of alginate 1 % w/v, photoinitiator 0.1% w/v and GelMA 3 % w/v or 6 % w/v. The polymeric mixture was vortexed for 30 seconds and centrifuged at 1,000 rpm to remove all the bubbles. Hydrogels were prepared in 24 well plate by casting the solution (0.5 ml) before UV irradiation for 2 minutes at nm 7 mw/cm2. Additionally, the polymeric mixture was used to coat prefabricated scaffolds of polycaprolactone (PCL) (1 cm x 1 cm) by soaking the scaffold in the solution for 1 minute before UV irradiation. To further reinforce the photo-crosslinked hydrogels, a further step of physical crosslinking was performed by soaking the hydrogels for 4 hours in solutions of CaCl 2. Different CaCl 2 concentrations were tested varying from 1 mm up to 10 mm. Control gels without any GelMA were also prepared simply by mixing the Alginate solution 2.5% with CaCl 2 1 mm. Polydopamine coating and dexamethasone adsorption studies IPN hydrogels were treated with dopamine in Tris-HCl buffer 10 mm (ph = 8.5) for 1 hour at 37 C and shaking at 60 rpm. Different dopamine concentrations were tested ranging from 0.5 mg/ml up to 2.0 mg/ml. Then, the hydrogel was washed with DPBS buffer to remove the excess of dopamine from the surface. S-7

8 A solution of Dex 0.1 mm in Tris buffer 10 mm was added on top of the coated hydrogel for 12 hours to ensure the adsorption of the drug to the pda layer. As a control, hydrogels were also soaked in Dex 0.1 mm solution without any pda treatment. Subsequently, the hydrogels were frozen at -80 C, freeze-dried and sputter coated with gold. The elementary composition of the hydrogel s surface was characterized by energy-dispersive X-ray (EDX) spectroscopy to evaluate the adsorption of Dex. To verify the retention of Dex on the hydrogel coated with pda, 200 µl a solution of Dex-FITC (7.75 µg/ml) was placed on the gels fabricated on the bottom of a 96 well plate (100 µl/well). As a control, the same study was carried out on gels without any pda coating. The fluorescence intensity of Dex-FITC was measured before and after the process of adsorption. The experiment was conducted for one hour in the dark at 37 C. The fluorescence was evaluated by using an excitation wavelength of 493 nm and by recording the emission wavelength at 519 nm. Dex-FITC was chosen instead of Dex to avoid the interference caused by the absorption of Irgacure 2959 in the UV region. Once the amount of Dex-FITC has been evaluated, the hydrogels were washed every 24 hours with 200 µl of fresh DPBS, and the fluorescence was monitored over a period of 120 hours. The quantity of Dex-FITC remaining on the gels was indirectly calculated based on the fluorescence intensity measured from the different washes and determined by a calibration plot of Dex-FITC in DPBS ranging from 0.39 µg/ml up to 25 µg/ml. Mechanical analysis of the hydrogels: The compressive modulus of the nanocomposite hydrogels was evaluated using an RSA-III dynamic mechanical analyzer (TA Instruments, New Castle, DE) and assessed under unconfined uniaxial compression with a 35 N load cell (n=5). The diameter of the gels was measured using a caliper, while the height was obtained directly using the RSA-III. All mechanical testing was performed on the swollen hydrogels, and the compression probes were lubricated with mineral oil both to minimize gel drying during the test. A compression rate of mm/s, corresponding to an average of 15% per min, was used. The compressive elastic modulus (E) S-8

9 was measured using the slope of the stress versus strain curve at the 10% strain. Samples were compressed unconstrained to 95% of their original height or to fracture point that was measured directly with the RSA-III. Viscosity measurements were carried out to evaluate the optimal viscosity of the polymeric solutions before UV irradiation. The tests were carried out using a cone-plate geometry (cone with 20 mm diameter, 1 angle) in the range of shear rate varying from 0.01 to 1000 s -1. The polymer alginate was solubilized in water, and the solutions were equilibrated at room temperature prior to the experiment. The study was conducted at the temperature of 25 C (n = 3). Frequency sweep in the range from 0.01 up to 10 Hz were recorded for all samples at 37 C in the viscoelastic region at 1% of strain. Preliminary strain sweep tests were carried out in the range of 0.1 up to 100% of strain to define the range of viscoelastic region. The hydrogels with the different concentration of GelMA or different concentration of calcium chloride (0.1 mm up to 10 mm) (1.0 cm in diameter and 0.5 cm height, n=3) were tested using a 25 mm serrated steel plate-plate geometry, and a water trap was placed on the geometry to avoid excessive evaporation during the test. Strain sweeps in the range of 1 to 300% of strain were carried out at 1 Hz to evaluate the maximum yield strain (n=3). Hydrogels were swollen before the study for 1 hour in PBS ph 7.4, and each study was repeated three times. Hydrogel physical characterization: Freeze-dried hydrogels were weighed and soaked in PBS at 37 C. The equilibrium swelling was evaluated by weighing the swollen hydrogel at different time points, and the swelling ratio (%) was calculated using the following equation: (%)= 100 where W s is the weight of the swollen hydrogel, and W d represents the weight of the freeze-dried hydrogel. In addition, to evaluate the hydrogel porosity and confirm the process of coating, the PCL S-9

10 scaffolds were characterized by scanning electron microscopy (SEM). Samples were mounted on a holder with double-sided conductive carbon tape and sputter-coated with gold. SEM images were obtained at an acceleration voltage ranging from 1 to 10 kv with an in-lens detector. To quantify the mean pore size image J analysis was carried by evaluating the size of 8 pores from 5 different pictures for each group tested. Results were reported as a mean ± standard deviation. Cell culture and biocompatibility studies: Passages 3-5 were used for all the studies. Additionally, human umbilical vein endothelial cells (HUVECs) were cultured in endothelial basal medium (EBM-2, Lonza) with complete endothelial growth supplements at standard culture conditions. Passage 3-6 were used for all the studies. To assess hydrogel biocompatibility, the hydrogels were fabricated in 24 well plate (0.5 ml/well) and soaked with different concentration of CaCl 2 (1 ml) ranging from 1 mm to 10 mm. Gels without any calcium were also tested as a control. The gels were washed overnight with PBS containing 1% of penicillin/streptomycin and soaked in complete EGM-2 growth media for 1 hour. 50 x 10 3 HUVECs were seeded on each gel and cells were allowed to grow for 24 hours. MTS colorimetric assay (λ =490 nm) was used to evaluate the cell viability and assess the hydrogel cytotoxicity. Data were calculated as the average of five different samples for each group tested. In addition, HUVECs morphology was evaluated at 24 hours after seeding by calcein staining in DPBS. Similarly, to investigate the biocompatibility of the dopamine coating, the gels were fabricated in 24 well plate (0.5 ml/well) and soaked with different concentration of dopamine for 1 hour at 37 C (1 ml) ranging from 0.5 up to 2.0 mg/ml. Gels without any pda treatment were used as a control. The gels were washed for 12 hours with PBS containing 1% of penicillin/streptomycin and soaked in α- MEM with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C and 5% CO 2. Then, 50 x 10 3 hascs were seeded on each gel and cells were allowed to grow for 48 hours. MTS colorimetric assay (λ =490 nm) was used to quantify cell viability and assess the hydrogel S-10

11 cytotoxicity. Data were reported as the average of five different samples for each group tested. In addition, hascs morphology was evaluated at 48 hours, and seeded gels were treated with a solution 0.1% Triton X-100 for 20 minutes followed by staining with Alexa Fluor 488 phalloidin, for imaging cell cytoskeleton, and DAPI to stain cell nuclei. Fluorescent images were taken using a fluorescent microscope (Zeiss). Differentiation studies of hascs: Human adipose-derived stem cells (hascs) were purchased from RoosterBio (USA) and maintained in Invitrogen alpha-minimum Essential Medium (α-mem) with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C and 5% CO2. Five different groups were tested. Group 1: Ctrl (-). hascs cultured on gel without any treatment in culture medium without any osteogenic factor (α MEM, 15% FBS, 1% Penicillin/Streptomycin). Group 2: Dex. hascs cultured on gel soaked with Dex 0.1 mm cultured in non-osteogenic medium (α MEM, 10% FBS, 1% Penicillin/Streptomycin 50 µm 2-phosphate ascorbic acid, 10 mm β- glycerophosphate, without Dex). Group 3: pda/dex. hascs cultured on gel pre-coated with a layer of pda and soaked afterward with Dex 0.1 mm. Cells were cultured in the same condition as the group 2. Group 4: Ctrl (+). hascs cultured on gel without any treatment in osteogenic medium (α MEM, 10% FBS, 50 µm 2-phosphate ascorbic acid, 10 mm β-glycerophosphate, with Dex 10 nm). Group 5: Ctrl (+) pda. hascs cultured on gel pre-coated with a layer of pda and cultured in osteogenic medium as group 4. Group 2 and 3 were necessary to study the effect of Dex with or without the pda layer. Group 4 and 5 are necessary to study the effect of pda in osteogenic medium. In all the systems, 50 x 10 3 hascs were seeded on the surface of the gels and were allowed to grow in α-mem with 15% fetal bovine serum (FBS) for 48 hours up to confluency. Then, the culture conditions were changed for the pda/dex and the Dex groups by adding to the media 50 µm 2- phosphate ascorbic acid, and 10 mm β-glycerophosphate. On the contrary, the Ctrl+ and the Ctrl+ pda groups were treated with osteogenic media (50 µm 2-phosphate ascorbic acid, 10 mm β- S-11

12 glycerophosphate, and 10 nm Dex). Media was changed every other day for the entire duration of the study. At 7 and 14 gels were stained for the presence of ALP following the standard protocol provided by the manufacturer (Alkaline Phosphatase kit, Sigma Aldrich, USA). ALP activity was measured at day 7 and day 14 using a fluorimetric assay (Abcam, catalog number: ab83371). hascs were detached from the surface of the hydrogels using 250 U/mL collagenase type IV at day 7 and 14 of cell culture. Cells were centrifuged for 5 minutes at 1,500 rpm, and the supernatant was discarded. Cells were homogenized in ALP buffer following the steps reported in the protocol provided by the manufacturer. Fluorescence was measured using an excitation of 360 and an emission wavelength of 440 nm in a microplate reader (Epoch, Biotek, USA). Five different samples for each group were tested to quantify the amount of ALP. Additionally, Alizarin Red staining was evaluated at 14 and 21 days, cells were fixed in 4% paraformaldehyde, washed with water three times, then incubated with 2% Alizarin Red (Sigma) for 20 minutes. Bright field images of the stained samples were taken after washing with water three times to remove any excess of staining. Calcium quantification was carried out at day 14 and day 21 using a colorimetric calcium quantification kit (Cayman, catalogue number: ) following the instruction provided by the manufacturer. Cells were detached from the gels surface using collagenase 250 U/mL and centrifuged at 1,000 g for 10 minutes. Cells lysate were obtained using the buffer provided by the kit, and five different samples for each group were analyzed using the colorimetric kit. Fabrication of PCL mesh constructs: The meshes were designed using Autodesk Inventor 2015 and exported to the STL file format (Figure S3). This was imported to Slic3r software to generate a code file that controls the printer movements. The meshes were printed on a RepRapPro Mendel 3D printer using a polycaprolactone (PCL) filament ( 99% purity, Makerbot Flexible Filament by Perstorp Polyols Inc,). The 1.75 mm filament was extruded through a 0.5 mm nozzle at 140 C. Struts for 30x30 mm meshes were extruded onto a glass plate to 500 µm width with a gap of 300 S-12

13 µm between them. The meshes were consisting of two layers of struts with an angle of 90 between the two layers, at a layer thickness of 500 µm. Statistical analysis: Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests used to determine whether a significant difference exists between specific groups. All statistical analyses were carried out with GraphPad Prism Software 6. A p-value less than 0.05 indicates statistical significance, which was displayed as * = p < 0.05, ** = p < 0.01, *** = p < S-13