Abstract
Due to its ubiquity and versatility in the human body, collagen is an ideal base material for tissue-engineering constructs. Chemical crosslinking treatments allow precise control of the biochemical and mechanical properties through macromolecular modifications to the structure of collagen. In this work, three key facets regarding the collagen crosslinking process are explored. Firstly, a comparison is drawn between the carbodiimide-succinimide (EDC-NHS) system and two emerging crosslinkers utilising alternate chemistries: genipin and tissue transglutaminase (TG2). By characterising the chemical changes upon treatment, the effect of EDC-NHS, genipin and TG2 crosslinking mechanisms on the chemical structure of collagen, and thus the mechanical properties conferred to the substrate is explored. Secondly, the relative importance of mechanical and biochemical cues on cellular phenomena are investigated, including cell viability, integrin-specific attachment, spreading and proliferation. Here, we observe that for human dermal fibroblasts, long-term, stable proliferation is preconditioned by the availability of suitable binding sites, irrespective of the substrate modulus post-crosslinking. Finally, as seen in the graphical abstract we show that by choosing the appropriate crosslinker chemistries, a materials selection map can be drawn for collagen films, encompassing both a range of tensile modulus and fibroblast proliferation which can be modified independently. Thus, in addition to a range of parameters that can be modified in collagen constructs, we demonstrate a route to obtaining tunable bioactivity and mechanics in collagen constructs is uncovered, that is exclusively driven by the crosslinking process.
Highlights
Collagen-I, a primary protein component of the extracellular matrix (ECM) has been widely employed as a base material for tissue engineering constructs with the aim of providing a similar chemical environment to the one experienced in vivo [1,2,3]
The crosslinking conditions are represented with respect to a standard crosslinker solution concentration: the ‘100%’ standards for EDCNHS, genipin and TG2 are defined in the Materials and Methods (Section 2)
Representative Fourier-transform Infrared (FTIR) spectra and triple helical contents measured for the non-crosslinked, EDC-NHS, genipin- and TG2 treated collagen films revealed no statistically significant differences in the triple helical content of the protein, indicating that minimal changes were imparted to the conformation of the protein upon crosslinking (Supplementary Information)
Summary
Collagen-I, a primary protein component of the extracellular matrix (ECM) has been widely employed as a base material for tissue engineering constructs with the aim of providing a similar chemical environment to the one experienced in vivo [1,2,3]. The ubiquity of collagen in connective tissue has promoted its use in medical devices tailored for a broad range of applications These include injectable hydrogels for cartilage repair [4] and dense fibrillar matrices prepared from neutralised acid-soluble collagen for dermal wound healing [5], collagen membranes as a periodontal barrier [6] and freeze-dried porous scaffolds fabricated from insoluble microfibrillar collagen for exvivo platelet production in flow bioreactors [7]. Though they vary in the type, source or solubility of collagen, these constructs can be crosslinked to tune their mechanical properties to match the loads experienced in-service at the host tissue site. To mimic these natural crosslinks, amine-based crosslinkers [14,15,16,17,18] have been considered to be ideal candidates to crosslink processed collagen in tissue engineering constructs
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