3 Methods
3.1 GM-HA Synthesis
1. Dissolve 1 g of hyaluronic acid in 100 mL of acetone/water
(50/50) solution overnight to prepare a 1% w/v solution.
2. Add 7.2 mL TEA (20-fold molar excess) until thoroughly
mixed (see Note 1).
3. Add 7.2 mL GM (20-fold molar excess) until thoroughly
mixed (see Note 2).
4. Stir overnight at room temperature (see Note 3).
5. Dialyze the resulting solution against DI water with 3.5 kDa
tubing at room temperature for 48 h (see Note 4).
6. Collect and freeze the dialyzed solution overnight at À80
C
(see Note 5).
7. Lyophilize the frozen solution for 48 h at 0.04 mbar and
À50
C.
8. Store the lyophilized GM-HA at À80
C for future use.
3.2 GelMA Synthesis
1. Dissolve 10 g of porcine skin gelatin in 100 mL DPBS by
stirring at 60
C to prepare a 10% w/v solution (see Note 6).
2. Add 8 mL of MA to the solution at a rate of 0.5 mL/min (see
Note 7).
3. Allow the reaction to continue for 3 h at 60
C with constant
stirring.
4. Add an equivalent volume of warm DPBS to dilute the resulting solution (see Note 8).
5. Dialyze the diluted solution against DI water with 13.5 kDa
tubing for 1 week at 40
C (see Note 9).
6. Collect and freeze the dialyzed solution overnight at À80
C
(see Note 5).
7. Lyophilize the frozen solution for 48 h at 0.04 mbar and
À50
C.
8. Store the lyophilized GelMA at À80
C for future use.
3.3 LAP Synthesis
1. All the following steps are performed in a chemical fume hood.
2. Add 3.2 g of 2,4,6-trimethylbenzoyl chloride dropwise to an
equal molar amount of dimethyl phenylphosphonite (3 g) with
constant stirring under argon.
3. Allow the reaction to continue for 18 h.
4. Dissolve 6.1 g lithium bromide to 100 mL of 2-butanone and
add into the previous mixture at fourfold excess.
5. Heat the reaction to 50
C until a solid precipitate forms in
about 10 min.
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