Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls

Abstract

Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physicochemical material properties. In this contribution, H-C-HSQC NMR spectra are used to gain detailed structural information on GMs and of 2-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the H NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simpleH NMR spectroscopy with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.

Beyond the Modification Degree: Impact of Raw Material on Physicochemical Properties of Gelatin Type A and Type B Methacryloyls

“…GM was prepared as previously described and the procedure was the same for G A and G B . GM derivatives were prepared with twofold (GM2), fivefold (GM5), and tenfold (GM10) molar excess of MAAnh with respect to a nominal amino group content of 0.35 mmol g −1 (described by van den Bulcke et al …”

“…NMR samples were prepared by dissolving ≈15 mg biopolymer in 600 µL D 2 O with TMSP as internal standard (1 mg mL −1 ). The DM was determined from 1 H NMR spectra from a Bruker Avance 500 spectrometer (500 MHz, room temperature), the DA was quantified from 1 H 13 C HSQC (heteronuclear single quantum coherence)‐spectra from a Bruker Avance‐III spectrometer (800 MHz, 37 °C) as reported previously by our group …”

Photoinduced Cleavage and Hydrolysis of o‐Nitrobenzyl Linker and Covalent Linker Immobilization in Gelatin Methacryloyl Hydrogels

“…[57] The DM was determined using the TMSPmethod, which uses the internal NMR-standard TMSP to calculate the amount of methacryl groups tethered to the gelatin upon functionalization. [57] The DM was determined using the TMSPmethod, which uses the internal NMR-standard TMSP to calculate the amount of methacryl groups tethered to the gelatin upon functionalization.…”

“…[57] The DM was determined using the TMSPmethod, which uses the internal NMR-standard TMSP to calculate the amount of methacryl groups tethered to the gelatin upon functionalization. [57] The DM amounted as follows: GM2A8 0.379 ± 0.013 mmol g −1 (n = 3); GM10 0.958 ± 0.068 mmol g −1 (n = 3); the total degree of modification was determined to be 0.797 ± 0.006 mmol g −1 (n = 3) for GM2A8, for GM10 the total modification degree equals the degree of methacryloylation. The DM is then calculated by comparing the acrylic protons integral with the integral of TMSP as recently introduced by our group.…”

“…[57] In short, a defined amount of TMSP is added to biopolymer samples with known concentration prior to 1 H-NMR measurement. [57] [57] The DM amounted as follows: GM2A8 0.379 ± 0.013 mmol g −1 (n = 3); GM10 0.958 ± 0.068 mmol g −1 (n = 3); the total degree of modification was determined to be 0.797 ± 0.006 mmol g −1 (n = 3) for GM2A8, for GM10 the total modification degree equals the degree of methacryloylation.…”