Dynamic Coordination Chemistry Enables Free Directional Printing of Biopolymer Hydrogel

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1 Supporting Information Dynamic Coordination Chemistry Enables Free Directional Printing of Biopolymer Hydrogel Liyang Shi, Hauke Carstensen, Katja Hölzl, Markus Lunzer, Hao Li, Jöns Hilborn, Aleksandr Ovsianikov, Dmitri A. Ossipov*, Division of Polymer Chemistry, Department of Chemistry-Ångström and Department of Physics and Astronomy, Uppsala University, Uppsala, 75121, Sweden. Institute of Materials Science and Technology, Technische Universität Wien, 1060 Vienna, Austria. Austrian Cluster for Tissue Regeneration, Austria. Department of Surgical Sciences, Uppsala University, Uppsala, 75185, Sweden Results HA-BP was synthesized in two steps (Figure S1A). Firstly, thiolated HA (HA-SH) was synthesized by carbodiimide (EDC)-mediated coupling of disulfide linker 1 to carboxylate groups of native HA, followed by in situ reduction of the disulfide bonds with dithiothreitol (DTT). The structure of HA-SH (degree of SH substitution ~8%) was verified by the appearance of respective signals at 2.81 and 2.68 ppm (-CH 2CH 2SH) in 1 H-NMR spectrum (Figure S1D). Secondly, the HA-BP derivate was obtained using thiol-ene addition reaction between HA-SH and acrylated BP reagent 2 (Figure S1A). 1H-NMR analysis of HA-BP revealed the appearance of a signal at 2.18 ppm, corresponding to protons of a methylene group adjacent to the BP bridging carbon (-CH 2C(OH)(PO 3H 2) 2). Additionally, successful grafting of acrylated BP 2 to HA backbone was confirmed by a single phosphorus signal (18.27 ppm) in the corresponding 31P-NMR spectrum (Figure S1B). Degree of bisphosphonate substitution in HA-BP was ~25% which corresponded to the attachment of approximately three BP groups to one thiol-terminated side chain of HA-SH precursor.

2 Figure S1. (A) Synthesis of HA-BP from thiol-modified HA (HA-SH). 1 H-NMR spectrum of the synthesized HA-BP. (B) 31 P-NMR spectrum of HA-BP. (C) 1 H-NMR spectrum of native HA and (D) 1 H-NMR spectrum of thiol-modified HA (HA-SH).

3 Figure S2. (A) FTIR spectra of acrylated pamidronate reagent 2 and its calcium salt. The last one had low solubility in water and precipitated from the solution (images on the right from FTIR spectra) due to binding of Ca 2+ ions to P-O - groups of pamidronate moiety. This binding was characterized by a shift of the phosphonate peak from 1085 cm -1 to 1066 cm -1. (B) FTIR spectra of HA-SH derivative and its calcium salt showing no change of IR bands of HA-SH derivative.

4 Figure S3. (A) The printable hydrogels were formed by simple mixing of HA-BP and CaCl 2 solutions. Higher biopolymer and ions concentrations favored hydrogel formation. (B) HA-BP Ca 2+ hydrogel disassembled upon incubation in PBS at acidic ph. Figure S4. After preparation of HA-BP Ca 2+ hydrogels, they were incubated in different media for 1 hour and their mechanical properties were evaluated by rheology. (A) Frequency sweep experiments showing storage modulus Gʹ (red curves) and loss modulus Gʹʹ (blue curves) of the hydrogels after incubation in 10 mm PBS at ph 7.4 (square symbols) and ph 6.0 (triangle symbols). (B) Frequency sweep experiments showing Gʹ and Gʹʹ of the hydrogels after incubation in 100 mm PBS, DMEM medium, 10 mm PBS, and water at ph 7.4 (listed in the order of decreasing of absolute values of the hydrogels moduli).

5 3D printer modification. To realize bioink extrusion, the standard extrusion parts of the printer for plastics were replaced with a syringe extruder. A syringe holder was designed to mount a hamilton microliter syringe directly on the movable printing platform. On top of the holder, a stepper motor was fixed. This motor was powered and controlled by the 3D printer. The syringe holder was designed to be as light as possible and steady enough to hold the syringe with high precision. The components of the modified printing head are shown in Figure S5. The syringe holder held the micro syringe at the shaft and at the top of the syringe. At the top of the holder, a stepper motor was fixed by four screws. The stepper motor moved a rod up and down. The rod was connected to a small clamp at its end that could move up and down thus pushing the syringe plunger. A cylinder at the bottom of the holder helped to fit it to the printing platform and centered it. A clamp was used to fix the holder to the platform. It allowed a quick mounting and unmounting of the holder while offering high stability. The syringe holder, the rotor clamp, the syringe clamp and the base clamp were designed in FreeCAD, a free open source parametric 3D modelling program (Figure S5B). The virtual 3D object was exported as stl-file which described the three dimensional shape by a triangulated closed surface. This file was imported into Cura, a free program that created the instructions for the 3D printer as a so called G-code file. The main part of the G-code consisted of 3D coordinates which described the motion path of the 3D platform and the amount of filament extruded per time unit. The holder was created by a Delta printer from a mixture of polylactide (PLA) and polyhydroxyalkanoate (PHA). The print was done layer by layer, and the object consisted of a shell and a filling. For the syringe holder, the shell thickness was 0.8 mm and the filling was a cubic grid with a density of 10% volume, ensuring a low weight holder (around 30 g).

6 Figure S5. Components of the modified 3D printing head: (A) A syringe pump is mounted directly on the printing platform of a 'Delta Tower' 3D printer. (B) The custom-built syringe mount is 3D printed. The outer ring of the shell is red, the inner ring is green, the infill is yellow, support structure is light blue and retraction motion is dark blue.

7 Figure S6. Hyaluronic acid dually functionalized with acrylamide (blue) and bisphosphonate (red) groups was synthesized from HA-BP derivative using EDC coupling. 1 H-NMR spectrum of the synthesized hyaluronic acid conjugated with acrylamide and bisphosphonate groups (Am-HA-BP).

8 Figure S7. Images of hydrogels with spatially defined immobilization of RGD peptide taken before incubation as well as after 1 and 3 days of incubation in PBS buffer. It is noteworthy that the distribution of fluorescent peptide molecules did not changed.