Cross-linkable alginate-graft-gelatin copolymers for tissue engineering Applications

G.-J. Graulus,a A. Mignon,a S. Van Vlierberghe,a,† H. Declercq,b K. Fehér,c M Cornelissen,b J.C. Martinsc

and P. Dubruela,†

Supporting information

Contents

Contents.................................................................................................................................................1

ATR-FT-IR spectroscopy..........................................................................................................................21H-NMR spectra......................................................................................................................................3

Thermogravimetric Analyses..................................................................................................................6

OPA analyses..........................................................................................................................................7

SEC analysis............................................................................................................................................8

Rheological evaluation of alginate-gelatin blends..................................................................................9

Cell adhesion assay...............................................................................................................................10

ATR-FT-IR spectroscopy

Figure S1: FT-IR spectra for the various materials

TABLE S1. Overview of the dominant absorption bands observed in ATR-FT-IR analysis.

Occurrence of the absorption bands (cm-1) in:

Vibration Gel-MOD Alginate Alg-gel(2%)

Alg-gel(4%)

Alg-gel(8%)

H-bond 3285 3248 3289 3292 3292COO-H 2941 2930 2936 2940 2940Amide I 1628 - 1630 1629 1629COO- anion - 1594 - - -Amide II 1534 - 1537 1543 1540δβ(OH) 1398 1406 1407 1403 1402Amide III 1234 - 1235 1237 1236υ(C-OH) 1031 1024 1031 1031 1031C1-H in α-pyranose - 882 876 876 -

1H-NMR spectra

Figure S2: 1H-NMR spectrum of alginate

Figure S3: 1H-NMR spectrum of Alg-gel 2%

Figure S4: 1H-NMR spectrum of Alg-gel 4%

Figure S5: 1H-NMR spectrum of Alg-gel 8%

Figure S6: 1H-NMR spectrum of Gel-MOD

Thermogravimetric Analyses

Figure S7: TGA Thermogram showing the decomposition of the various samples. Graph shows alginate (black points), Alg-gel 2% (red, dash point), Alg-gel 4% (blue, short dash), Alg-gel 8% (green dashes) and Gel-MOD (dark cyan, solid). The gelatin content dominates the decomposition profile, making it difficult to correlate the decomposition steps to the chemical composition of the samples.

OPA analyses

Figure S8: Calibration curve obtained for n-butylamine following the OPA protocol. Linear fitting results in the following relation: y = 110.58x+0.1227 (R2=0.9907)

Table S2: determination of the number of consumed amines during the grafting protocol. Results show 71-82% decrease in amine content.

Material Alg-gel 2% Alg-gel 4% Alg-gel 8% Gel-MOD

Sample mass [mg] 1.25 1.25 1.25 1.25

Mass percentage gel-MOD [%] 70.55 79.09 88.32 100.00

Mass gel-MOD in sample [mg] 0.88 0.99 1.10 1.25

Number of amines available[mmol] 1.01E-04 1.14E-04 1.27E-04 1.44E-04

Relative absorbance 1.73E-01 1.94E-01 1.73E-01 4.40E-01

Standard deviation 1.36E-02 9.53E-03 8.07E-03 2.38E-02

Concentration of amines [M] 4.54E-04 6.42E-04 4.54E-04 2.87E-03

Number of amines in sample volume [mmol] 2.27E-05 3.21E-05 2.27E-05 1.44E-04

Number of reacted amines [mmol] 7.86E-05 8.15E-05 1.04E-04 -

Effectiveness [%] 77.60 71.72 82.11 -

SEC analysis

Figure S9: SEC traces showing the increase in molecular weight when gelatin is grafted on alginate.

TABLE S3. SEC results for Gel-MOD after incubation in acetate buffer (pH 3.4) for 24 hours. The results show a clear reduction in molecular weight as compared to virgin Gel-MOD.

Material Mn [Da] Mw [Da] Đ

Gel-MOD prior to incubation 81680 235500 2.88

Gel-MOD after to incubation 50430 188460 3.74

Rheological evaluation of alginate-gelatin blends

Figure S10: Storage moduli obtained for representative blends of modified gelatin and alginate at 21°C (white bar) and at 40°C (grey bar). The results were found to be an order of magnitude lower than comparable alginate-graft-gelatin copolymers.

Cell adhesion assay

Figure S11: Percentage of viable HFF-1 cells cultured on hydrogel films (Alg-Gel 2%, 4% and 8% activation; Gel-MOD) for 1 day relative to the control (tissue culture polystyrene) The amount of viable cells was quantified with the MTT-assay. Significant differences (p>0.05) are indicated with an asterisk (*).

Figure S12: Photographs of HFF-1 cells cultured on hydrogel films (Alg-Gel 2% (row 1); Alg-Gel 4% (row 2); Alg-Gel 8% (row 3); Gel-MOD (row 4) and TCPS (row 5) after 1 day. Scale bars represent 500 µm (left column) or 200 µm (right column). Fluorescent micrographs after live/dead staining.