Thiol-ene photo-click hydrogels were prepared via step-growth polymerisation using thiol-functionalised type-I collagen and 8-arm poly(ethylene glycol) norbornene-terminated (PEG-NB), as a potential injectable regenerative device. Type-I collagen was thiol-functionalised by a ring opening reaction with 2-iminothiolane (2IT), whereby up to 80 Abs. % functionalisation and 90 RPN% triple helical preservation were recorded via 2,4,6-Trinitrobenzenesulfonic acid (TNBS) colorimetric assay and circular dichroism (CD). Type, i.e., either 2-Hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (I2959) or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and concentration of photoinitiator were varied to ensure minimal photoinitiator-induced cytotoxicity and to enable thiol-ene network formation of collagen-PEG mixtures. The viability of G292 cells following 24 h culture in photoinitiator-supplemented media was largely affected by the photoinitiator concentration, with I2959-supplemented media observed to induce higher toxic response (0.1 → 0.5% (w/v) I2959, cell survival: 62 → 2 Abs. %) compared to LAP-supplemented media (cell survival: 86 → 8 Abs. %). In line with the in vitro study, selected photoinitiator concentrations were used to prepare thiol-ene photo-click hydrogels. Gelation kinetics proved to be largely affected by the specific photoinitiator, with LAP-containing thiol-ene mixtures leading to significantly reduced complete gelation time (τ: 187 s) with respect to I2959-containing mixtures (τ: 1683 s). Other than the specific photoinitiator, the photoinitiator concentration was key to adjusting the hydrogel storage modulus (G'), whereby 15-fold G' increase (232 → 3360 Pa) was observed in samples prepared with 0.5% (w/v) compared to 0.1% (w/v) LAP. Further thiol-ene formulations with 0.5% (w/v) LAP and varied content of PEG-NB were tested to prepare photo-click hydrogels with porous architecture, as well as tunable storage modulus (G': 540-4810 Pa), gelation time (τ: 73-300 s) and swelling ratio (SR: 1530-2840 wt %). The photoinitiator-gelation-cytotoxicity relationships established in this study will be instrumental to the design of orthogonal collagen-based niches for regenerative medicine.
Currently two factors hinder the use of collagen as building block of regenerative devices: the limited mechanical strength in aqueous environment, and potential antigenicity. Polymeric collagen is naturally found in the cross-linked state and is mechanically tougher than the monomeric, acid-soluble collagen ex vivo. The antigenicity of collagen, on the other hand, is mainly ascribed to inter-species variations in amino acid sequences of the non-helical terminal telopeptides. These telopeptides can be removed through enzymatic treatment to produce atelocollagen, although the effect of this cleavage on triple helix organization, amino acidic composition and thermal properties is often disregarded. Here, we compare the structural, chemical and physical properties of polymeric and monomeric type I collagen with and without telopeptides, in an effort to elucidate the influence of either mature covalent crosslinks or telopeptides. Circular dichroism (CD) was used to examine the triple helical conformation and quantify the denaturation temperature (T) of both monomeric collagen (36.5°C) and monomeric atelocollagen (35.5°C). CD measurements were combined with differential scanning calorimetry (DSC) in order to gain insight into the triple helix-to-coil thermal transition and shrinkage temperature (T) of polymeric atelo collagen (44.8°C), polymeric collagen (62.7°C), monomeric atelo collagen (51.4°C) and monomeric collagen (66.5°C). Structural and thermal analysis was combined with high pressure liquid chromatography (HPLC) to determine the content of specific collagen amino acidic residues used as markers for the presence of telopeptides and mature crosslinks. Hydroxylamine was used as the marker for polymeric collagen, and had a total content of 9.66% for both polymeric and polymeric atelo collagen; tyrosine was used as the marker for telopeptide cleavage, was expressed as 0.526% of the content of polymeric collagen and the partially-reduced content of 0.39% for atelocollagen.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.