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Surpassing the mechanical boundaries of crosslinkable gelatin

Jasper Van Hoorick (UGent) , Heidi Declercq (UGent) , Maria Cornelissen (UGent) , Peter Dubruel (UGent) and Sandra Van Vlierberghe (UGent)
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Abstract
INTRODUCTION: The present work aims to develop gelatin hydrogels with tunable mechanical properties which reach beyond the capabilities of currently applied methacrylamide modified gelatin (gel-MOD) materials. To this end, both the primary amines as well as the carboxylic acids in the gelatin backbone will be functionalized with crosslinkable moieties followed by crosslinking via UV irradiation. MATERIALS AND METHODS: In a first part, the free amines present in gelatin B were functionalized using 2.5 equivalents of methacrylic anhydride (relative to the amount of free amines present in gelatin) in a phosphate buffered solution (pH 7.3) (10 w/v%) to obtain methacrylamide-modified gelatin (gel-MOD) with a degree of substitution (DS) of 97%. Additional crosslinkable functionalities were introduced by the reaction of the carboxylic acids in gel-MOD with amino-ethylmethacrylate (AEMA) (2 eq) via conventional carbodiimide coupling chemistry (EDC/NHS) in DMSO under inert atmosphere (DSCOOH 30%). Via film casting, 10 w/v% solutions of gel-MOD (DS 97%) and gel-MOD-AEMA containing 2 mol% of Irgacure 2959 were crosslinked by application of UV-light (365 nm) for 30 minutes. The obtained hydrogels were characterized using differential scanning calorimetry, gel fraction/swelling tests, rheology and (HR-MAS) 1H NMR spectroscopy. Finally, the biocompatibility was assessed via live/dead staining experiments. RESULTS AND DISCUSSION: Interestingly, the gel-MOD-AEMA hydrogel precursors could be solubilized at room temperature. To investigate this phenomenon DSC experiments where performed which displayed a drop in upper critical solution temperature (UCST) together with a significant reduction in the associated energy of this phase transition. Both gel fraction tests as well as HR-MAS 1H NMR measurements indicated a higher crosslinking efficiency for the AEMA derivatives. As anticipated, swelling tests revealed that the AEMA derivatives were characterized by poorer swelling properties compared to gel-MOD (450% vs 700%) originating from a higher network density. Real-time rheological monitoring of the crosslinking reaction further supported the envisaged concept. The gel-MOD-AEMA derivative exhibited a storage modulus roughly twice the value of the gel-MOD derivative (see figure below). Furthermore, live/dead staining experiments revealed a comparable biocompatibility for both derivatives. CONCLUSIONS: The incorporation of additional crosslinkable functionalities via reaction of the carboxylic acids present in gel-MOD with AEMA provides an excellent method to increase the structural integrity of hydrogel materials. Furthermore, both materials show a comparable biocompatibility thereby maintaining the tissue engineering potential. These results are promising towards the application of two-photon polymerization on gelatin hydrogels to yield the ideal, structurally and mechanically tailored hydrogel scaffold for soft tissue engineering. 1 REFERENCES 1. Ovsianikov, A. et al. Biomacromolecules 12, 851–858 (2011)
Keywords
gel-MOD-AEMA, Gelatin, hydrogel, gel-MOD, crosslinkable, methacrylamides, methacrylates

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Chicago
Van Hoorick, Jasper, Heidi Declercq, Maria Cornelissen, Peter Dubruel, and Sandra Van Vlierberghe. 2014. “Surpassing the Mechanical Boundaries of Crosslinkable Gelatin.” In AMBA 2014 : Advanced Materials for Biomedical Applications.
APA
Van Hoorick, J., Declercq, H., Cornelissen, M., Dubruel, P., & Van Vlierberghe, S. (2014). Surpassing the mechanical boundaries of crosslinkable gelatin. AMBA 2014 : advanced materials for biomedical applications. Presented at the Conference on Advanced Materials for Biomedical Applications (AMBA 2014).
Vancouver
1.
Van Hoorick J, Declercq H, Cornelissen M, Dubruel P, Van Vlierberghe S. Surpassing the mechanical boundaries of crosslinkable gelatin. AMBA 2014 : advanced materials for biomedical applications. 2014.
MLA
Van Hoorick, Jasper, Heidi Declercq, Maria Cornelissen, et al. “Surpassing the Mechanical Boundaries of Crosslinkable Gelatin.” AMBA 2014 : Advanced Materials for Biomedical Applications. 2014. Print.
@inproceedings{5801821,
  abstract     = {INTRODUCTION: The present work aims to develop gelatin hydrogels with tunable mechanical properties which reach beyond the capabilities of currently applied methacrylamide modified gelatin (gel-MOD) materials. To this end, both the primary amines as well as the carboxylic acids in the gelatin backbone will be functionalized with crosslinkable moieties followed by crosslinking via UV irradiation.
MATERIALS AND METHODS: In a first part, the free amines present in gelatin B were functionalized using  2.5 equivalents of methacrylic anhydride (relative to the amount of free amines present in  gelatin) in a phosphate buffered solution (pH 7.3) (10 w/v\%) to obtain methacrylamide-modified gelatin (gel-MOD) with a degree of substitution (DS) of 97\%. Additional crosslinkable functionalities were introduced by the reaction of the carboxylic acids in gel-MOD with amino-ethylmethacrylate (AEMA) (2 eq) via conventional carbodiimide coupling chemistry (EDC/NHS) in DMSO under inert atmosphere (DSCOOH 30\%). Via film casting, 10 w/v\% solutions of gel-MOD (DS 97\%) and gel-MOD-AEMA containing 2 mol\% of Irgacure 2959 were crosslinked by application of UV-light (365 nm) for 30 minutes. The obtained hydrogels were characterized using differential scanning calorimetry, gel fraction/swelling tests, rheology and (HR-MAS) 1H NMR spectroscopy. Finally, the biocompatibility was assessed via live/dead staining experiments.
RESULTS AND DISCUSSION: Interestingly, the gel-MOD-AEMA hydrogel precursors could be solubilized at room temperature. To investigate this phenomenon DSC experiments where performed which displayed a drop in upper critical solution temperature (UCST) together with a significant reduction in the associated energy of this phase transition. Both gel fraction tests as well as HR-MAS 1H NMR measurements indicated a higher crosslinking efficiency for the AEMA derivatives. As anticipated, swelling tests revealed that the AEMA derivatives were characterized by poorer swelling properties compared to gel-MOD (450\% vs 700\%) originating from a higher network density.  Real-time rheological monitoring of the crosslinking reaction further supported the envisaged concept. The gel-MOD-AEMA derivative exhibited a storage modulus  roughly twice the value of the gel-MOD derivative (see figure below). Furthermore, live/dead staining experiments revealed a comparable biocompatibility for both derivatives.  
CONCLUSIONS: The incorporation of additional crosslinkable functionalities via reaction of the carboxylic acids present in gel-MOD with AEMA provides an excellent method to increase the structural integrity of hydrogel materials. Furthermore, both materials show a comparable biocompatibility thereby maintaining the tissue engineering potential. These results are promising towards the application of two-photon polymerization on gelatin hydrogels to yield the ideal, structurally and mechanically tailored hydrogel scaffold for soft tissue engineering. 1
REFERENCES
1. Ovsianikov, A. et al. Biomacromolecules 12, 851--858 (2011)},
  author       = {Van Hoorick, Jasper and Declercq, Heidi and Cornelissen, Maria and Dubruel, Peter and Van Vlierberghe, Sandra},
  booktitle    = {AMBA 2014 : advanced materials for biomedical applications},
  language     = {eng},
  location     = {Ghent, Belgium},
  title        = {Surpassing the mechanical boundaries of crosslinkable gelatin},
  year         = {2014},
}