Supporting information for Biomacromolecules article:

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1 Supporting information for Biomacromolecules article: Self-Healing Dynamic Hydrogel as Injectable Shock-Absorbing Artificial Nucleus Pulposus by Adrián Pérez-San Vicente, Marianna Peroglio, Manuela Ernst, Pablo Casuso, Iraida Loinaz,* Hans-Jürgen Grande, Mauro Alini, David Eglin, Damien Dupin*

2 Materials and Methods Cell culture Bovine nucleus pulposus cells (NP cells) were cultured in medium containing DMEM (Sigma), 10% fetal bovine serum (Lonza), 1X non-essential amino acid (NEAA) (Sigma), 1% penicillin/ streptomycin (Sigma), 2mM L glutamine (Sigma). Trypsin (0.25%)/EDTA were used to harvest the cells at 80% confluency. Cell cultures were maintained at 37ºC in an incubator with 95% humidity and 5% of CO2. The cultures were replenished with fresh medium at 37ºC twice a week. Determination of cell viability and proliferation Cell Titer-Glo Luminescent assay The effect of the hydrogel on NP cell growth was evaluated using Cell titer-glo Lumininescent cell viability assay (Promega) up to 7 days. NP cells were seeded at a density of 1500 cells/ cm2 in 24 well plates and allowed to grow for 24 hours. After that period, approximately 100 µl of hydrogel were placed on seeded cultured NP cells and further incubated for 1, 3 and 7 days. As a proliferation control, NP cell culture media and Triton X- 100 were used. The medium was changed once 24 h after the incubation with cell culture inserts. At each time point cells were cultured for 10 minutes with a volume of Cell Titer-Glo reagent equal to the volume of cell culture medium present in each p24 well (e.g add 300 µl of Cell Titer-Glo Reagent to 300 µl of cell culture medium present in each well). The luminiscence per well was measured in opaque p96 well plates using a micro-plate reader. All experiments were performed in triplicate.

3 Live-Dead assay Cell viability after hydrogel co-culture was assessed with the Live-Dead assay (Invitrogen). NP cells were seeded at a density of 1500 cells/ cm2 in 22-mm diameter coverslips (Menzel- Glaser). Samples were processed according to the manufacturer s recommendation. At day 7 the cell culture inserts were removed from the 24 well culture plates and cells attached to the coverslips were rinsed with PBS1X (Gibco) and processed for Live-Dead assay. Cells were incubated with the ethidium-calcein mixture and incubated for 30 at room temperature. After this treatment, the slides were prepared for microscopy on glass slides by mounting with Vectashield (Vector). A fluorescence microscope (Leica, DM 2000) and associate software were used to visualize the viability of cells.

4 Figure S1. Digital picture showing one of the specimen plated with PMMA and ready to place in the PTS apparatus before mechanical testing. Form the right-hand side picture it can be clearly seen that the PMMA plates are held by the 3 points type circular holder of the MTS apparatus. Figure S2. Representative cyclic load displacement data. Load vs. time data showing that load is not varying significantly during the 5 cycles. Thus, the 5 th cycles was used to plot the force vs. displacement curve from Figure 3. Figure S3. Data analysis. Representative compression-tension curve with corresponding curve fits. Tensile stiffness and Compression stiffness are calculated from a linear regression of the load-displacement data between the 70% and 100% of maximum load. The neutral zone is calculated from a polynomial fit of all data, determining the inflection point from the minimum derivative. The slope equal to the derivative of the polynomial model (SNZ) extending through the inflection point was defined as neutral zone stiffness. Figure S4. (A) cell viability at day 1, 3 and 7 measured by Cell Glo Titer luminescent assay for healthy bovine NP cells alone (positive control), NP cells and DH and NP cells and triton X100 (negative control). (B) Optical microscope pictures of NP cells alone and in the presence of DH at day 1, 3 and 7. (C) Live/Dead assay showing the 3 groups at day 7 with cell alive in green and dead cells in red. Figure S5. Determination of mechanical parameters: Tensile and compressive stiffness Mean and standard deviations of compression and tensile stiffness. No significant differences in tensile and compressive stiffness were observed between groups. Figure S6. Transversal cuts with sharp blade of the IVDs specimen containing the hydrogels after mechanical testing showed that the NP cavity remained filled but the native tissue and the injected material were not clearly distinguishable.

5 Figure S7. Representative µct images and reconstructions of the NP-nucleotomized IVD before and after the entire mechanical testing. i.e. compression/tension at different frequency and creep tests. Figure S8. Box chart showing mechanical parameters of all samples (intact) The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values are represented in box chart minimum, median, mean, maximum. The box chart was generated using OriginPro8 software (n=7). Figure S9. Box chart showing mechanical parameters of all samples (dynamic) The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values are represented in box chart minimum, median, mean, maximum. The box chart was generated using OriginPro8 software (n=7). Figure S10. Box chart showing mechanical parameters of all samples (covalent) The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values are represented in box chart minimum, median, mean, maximum. The box chart was generated using OriginPro8 software (n=7). Figure S11. Box chart showing mechanical parameters of all samples (cavity) The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values are represented in box chart minimum, median, mean, maximum. The box chart was generated using OriginPro8 software (n=7).

6 Figure S1. Digital picture showing one of the specimen plated with PMMA and ready to place in the PTS apparatus before mechanical testing. Form the right-hand side picture it can be clearly seen that the PMMA plates are held by the 3 points type circular holder of the MTS apparatus.

7 Figure S2. Representative cyclic load displacement data. Load vs. time data showing that load is not varying significantly during the 5 cycles. Thus, the 5 th cycles was used to plot the force vs. deformation curve from Figure 3.

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9 Figure S3. Data analysis. Representative compression-tension curve with corresponding curve fits. Tensile stiffness and Compression stiffness are calculated from a linear regression of the load-displacement data between the 70% and 100% of maximum load. The neutral zone is calculated from a polynomial fit of all data, determining the inflection point from the minimum derivative. The slope equal to the derivative of the polynomial model (SNZ) extending through the inflection point was defined as neutral zone stiffness.

10 Figure S4. (A) cell viability at day 1, 3 and 7 measured by Cell Glo Titer luminescent assay for healthy bovine NP cells alone (positive control), NP cells and DH and NP cells and triton X100 (negative control). (B) Optical microscope pictures of NP cells alone and in the presence of DH at day 1, 3 and 7. (C) Live/Dead assay showing the 3 groups at day 7 with cell alive in green and dead cells in red.

11 Figure S5. Determination of mechanical parameters: Tensile and compressive stiffness Mean and standard deviations of compression and tensile stiffness (n=7). No significant differences in tensile and compressive stiffness were observed between groups.

12 Figure S6. Transversal cuts with sharp blade of the IVDs specimen containing the hydrogels after mechanical testing showed that the NP cavity remained filled but the native tissue and the injected material were not clearly distinguishable.

13 Figure S7. Representative µct images and reconstructions of the NP-nucleotomized IVD before and after the entire mechanical testing. i.e. compression/tension at different frequency and creep tests.

14 Figure S8. Box chart showing mechanical parameters of intact discs The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values represented in box chart are minimum, median, mean, maximum. The box chart was generated using OriginPro8 software (n=7).

15 Figure S9. Box chart showing mechanical parameters of discs restored with dynamic hydrogel The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values represented in box chart are minimum, median, mean, maximum (n=7).

16 Figure S10. Box chart showing mechanical of discs restored with covalent hydrogel The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values represented in box chart are minimum, median, mean, maximum (n=7).

17 Figure S11. Box chart showing mechanical of partially nucleotomized discs The box is determined by the 25 th and 75 th percentiles. The whiskers are determined by the 5 th and the 95 th percentiles. Additional values are represented in box chart minimum, median, mean, maximum. The box chart was generated using OriginPro8 software (n=7). 17