Acrl peg sva

Addition of acryl groups to RGDS (American Peptide), human recombinant carrier free BMP2 (R&D systems), and human recombinant carrier free TGFβ1 (EMD Millipore) was carried out via reaction with acryloyl-PEG-succinimidyl valerate (ACRL-PEG-SVA, 3.4 kDa; Laysan Bio) at a 1:1 molar ratio for RGDS or 1:6 molar ratio for BMP2 and TGFβ1. Reconstituted TGFβ1 and BMP2 were first dialyzed to non-growth factor associated primary amines. The resulting mixtures were reacted for 2 hr in 50 mM sodium bicarbonate buffer, pH 8.5.^{74 ,75} The ACRL-PEG-RGDS was purified by dialysis overnight against dIH₂O at 4°C using 3500 MWCO Snakeskin Dialysis tubing (Thermo Fisher Scientific) to remove unreacted ACRL-PEG-SVA. ACRL-PEG-BMP2 and ACRL-PEGTGFβ1 were purified by dialysis overnight against dIH₂O at 4°C using 5000 MWCO Snakeskin Dialysis tubing.

PEGDA was prepared as previously described by combining 0.1 mmol mL⁻¹ dry PEG (10 kDa, Fluka), 0.4 mmol mL⁻¹ acryloyl chloride, and 0.2 mmol mL⁻¹ triethylamine in anhydrous dichloromethane with stirring under argon overnight.^{68 (link)} The resulting solution was washed with 2 M K₂CO₃ and separated into aqueous and dichloromethane phases to remove HCl. The dichloromethane phase was subsequently dried with anhydrous MgSO₄, and PEGDA was precipitated in diethyl ether, filtered, and dried under vacuum. The extent of PEG diacrylation was determined by ¹H NMR to be ~95%.
Recombinant human bFGF (17 kDa; 13256-029, Life Technologies) and the cell adhesion peptide RGDS (American Peptide) were reacted with acryloyl-PEG-N-hydroxysuccinimide (ACRL-PEG-SVA) (3.4 kDa, Laysan Bio) at a 1:3 and 1:1 molar ratio, respectively, for 2 h in 50 mM sodium bicarbonate buffer, pH 8.5.^{56 (link),69 (link)} Recombinant human bFGF has previously been shown to effectively interact with both murine^{70 (link)} and porcine^{71 (link)} cells. In addition, bFGF is known to maintain its potency following reaction with an acrylate-derivatized PEG linker and subsequent incorporation within a PEGDA hydrogel.^{72 (link)} ACRL-PEG-bFGF was purified by dialysis, stored at −20 °C, and used within 24 h of storage. ACRL-PEG-RGDS was purified by dialysis, lyophilized, and stored at −80 °C until use.

In order to covalently link the Scl2-2 protein into the structure of PEGDA hydrogels, the Scl2-2 protein was acrylate-derivatized using PEG-succinimidyl valerate (ACRL-PEG-SVA, 3.4 kDa; Laysan Bio). Briefly, Scl2-2 trimer and ACRL-PEG-SVA were reacted at a molar ratio of 1:6 for 2 h in phosphate buffered saline (PBS, pH 7.4; Life Technologies). Immediately following the derivation reaction, the resulting ACRL-PEG-Scl2-2 product was used for the fabrication of the PEG-Scl2-2 hydrogels. As a control, rat tail collagen I (Life Technologies) was also reacted with ACRL-PEG-SVA for 2 h at a 1:6 molar ratio in 50 mM sodium bicarbonate buffer, pH 8.5.^{70 (link)} The resulting products were then dialyzed against double deionized water for 48 h. Following purification, the acrylate-derivatized collagen was lyophilized and stored at -80 ºC until use. The incorporation of acrylate groups within the Scl2-2 and collagen I was confirmed by Fourier Transform Infrared Spectroscopy following previously described methods.^{57 (link)} Both acrylate-derivatized and unmodified Scl2-2 were also analyzed by circular dichroism as previously described.^{57 (link)} Results from the circular dichroism analyses are shown in Supplementary Figure 1.