Abstract
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.
Highlights
The design of regenerative devices relies heavily on the use of three-dimensional (3D) scaffolds to provide the appropriate environment, mechanical support and an initial cell anchorage site for the regeneration of tissues and organs [1]
We explored the challenge associated with the formation of an injectable, regenerative device via a thiol-ene photo-click reaction in the presence of 2-iminothiolane (2IT)-functionalised collagen and 8-arm norbornene-terminated poly(ethylene glycol) (PEG-NB). 2IT was selected as a flexible thiol-forming compound and was covalently-coupled to type I rat tail collagen via ring-opening reaction using the amino group fromlysine terminations
Sample nomenclature used in this work is as follows: thiol-ene hydrogels are coded as “CollPEGX”, where “Coll” and “PEG” refer to 2IT-functionalised collagen and 8-arm norbornene-terminated PEG, respectively, and “X” identifies the PEG content
Summary
The design of regenerative devices relies heavily on the use of three-dimensional (3D) scaffolds to provide the appropriate environment, mechanical support and an initial cell anchorage site for the regeneration of tissues and organs [1]. As a result of the reaction selectivity and oxygen insensitivity, an interesting feature of thiol-ene mixtures is their potential to be delivered as injectable liquid promptly leading to the formation of a tissue cavity-conformable hydrogel following application of a suitable external stimulus. To achieve collagen gelation in situ, a major hurdle for the thiol-ene photo-click reaction is the necessary use of a non-toxic and water-soluble photoinitiator [1,36]. The presence of NB-terminated multi-arms was expected to minimise reaction-hindering steric effects, thereby promoting faster gelation compared to linear PEG-NB variants.We hypothesised that the orthogonality offered by the thiol-ene reaction could result in the rapid formation of a defined collagen based hydrogel network (~seconds) and minimal toxic response [39]. Two water-soluble photoinitiators were employed to establish the photoinitiator-gelation-cytotoxicity relationship involved in the formation of thiol-ene photo-click collagen-PEG hydrogels
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