Tunable bioactivity and mechanics of collagen-based tissue engineering constructs: A comparison of EDC-NHS, genipin and TG2 crosslinkers

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.

Tunable bioactivity and mechanics of collagen-based tissue engineering constructs: A comparison of EDC-NHS, genipin and TG2 crosslinkers.  EDC-NHS crosslinks via an amide mechanism, making use of the glutamates (E) in the amino acid sequence. These glutamates form an integral part of the GFOGER metal ion-dependent adhesion sites in collagen for integrin specific binding. Unlike EDC-NHS, genipin and transglutaminase, bypass the use of aspartates and glutamates and crosslink solely via the primary amines. Where transglutaminase is unable to find suitable amines for crosslinking, glutamines and some existing amides are converted into glutamates (E).

Fourier-transform Infrared Spectroscopy
Representative FTIR spectra of the non-crosslinked, EDC-NHS, genipin-and TG2 treated collagen films are shown in Figure 2a. As seen in Figure 2b, no statistically significant differences are observed across the data sets in the triple helical or α-helical components of the protein, indicating little change to the triple helical conformation. The proportion of amide I peak occupied by the triple helical peak is also consistent with values obtained in the literature.

Representative stress-strain curves
Representative stress-strain curves of the collagen films under tension are illustrated in Figure 3. A typical curve presents four regions: an initial toe region, linear-elastic extension, plastic deformation and finally catastrophic failure. The toe region is present in all treated samples, although it is less pronounced in the highly crosslinked films, particularly Genipin at 200% and EDC-NHS 100%. Although all films also demonstrated limited plastic deformation prior to failure, a short plateau region was visible for the noncrosslinked and 60% genipin crosslinked films.

Quantitative cell characterisation
Cell area, perimeter, circularity, aspect ratio and roundness were subsequently measured for 10 cells in total across independent experiments performed in quadruplicate.
Circularity and round are shape parameters that can help delineate distinct stages of cell spreading characterised by cell polarisation by considering the relative effects of perimeter, area and aspect ratio. Roundness is a direct measure of the cell polarisation and aspect ratio, whereas circularity is an indirect measure of the peripheral smoothness by including the effect of contact perimeter. The data reveal no significant differences in the mean cell area, cell perimeter or circularity of the cells when comparing cells on treated substrates to the non-crosslinked condition. However, a significant increase in cell roundness is seen at 50% and 100% EDC-NHS, 100% and 200% genipin, and 200% TG2 when compared with the non-crosslinked condition. Although values can vary significantly between cell types, fully spread fibroblasts have been reported to have an area of 1250 µm 2 as opposed to the 300 µm 2 surface area of unspread rounded fibroblasts. 2 In comparison with these values, the higher cell area for all conditions indicates that the cells are well-spread on all conditions of crosslinking, actively maximise their area of contact with the substrate. The cell perimeter has been reported to represent both the affinity of the cell for a given surface but also can reflect different stages of cell spreading. 2   The expression of Collagen-I and GAPDH by the human dermal fibroblasts seeded on untreated and EDC-NHS, genipin-and TG2-treated collagen films are shown in Figure 6, where similar levels of expression are observed for cells seeded on all untreated and treated substrates.  Figure 7 indicates an increase in the protease secretion on all EDC-NHS treated substrates, the low crosslinking concentratons of Genipin (40%) and the high crosslinking concentrations of TG2 (100 and 200%). Although this study cannot conclusively identify the mechanisms by which the different crosslinkers regulate MMP1 expression, we speculate that the deviation in the expected glutamic acid residues, arising both with EDC-NHS and TG2 treatment, but not with genipin treatment, may result in the upregulation of MMP1 by the human dermal fibroblasts.

Swelling
In general, the treatments were not observed to affect the material significantly as seen in Figure 8, with the exception of a statistically significant decrease in water uptake at the highest crosslinking concentrations of genipin and TG2. As a result, the low swelling of the films when combined with their low thickness, little to no cell-infiltration is expected within the films and all cellular behaviour is likely to be limited to the surface of the films. It must also be noted that the trends observed here on the thin collagen films only reflect changes in hydrophilicity and are not comparable to 3D hydrogels or porous scaffolds where several networks for water infiltration are available. In such 3D constructs, other materials properties such as the mechanical strength will also play a role in the ability of the hydrogels to swell.

Photographs and Optical Micrographs
As seen in the films shown in Figure 9, all films are clear and colourless with the exception of genipin-treated films which are clear but blue in colour after crosslinking. In spite of the blue colour of genipin-crosslinked constructs, the crosslinker has been extensively investigated in vivo for its use in corneal implants. 3,4,5 No delamination or macroscopic shrinkage is observed on the films adhered to the well plates after 7 day cell culture on the films, indicating good stability of films during the cell culture period. Transmission phase contrast images of the films following 7 days of cell culture are also shown in Figure 10. At low crosslinking conditions (all TG2 and low EDC-NHS crosslinking), some shrinkage/ loss of material is observed around the edges of the films. Regardless, the central regions where cells were seeded and imaged are all observed to be well-adhered and unaffected over the 7-day cell culture period. Images were obtained using the Axiocam 503 Zeiss Fluorescence Microscope.
Non-crosslinked EDC-NHS Genipin TG2 Low Medium High Figure 10: Phase contrast micrographs of untreated, EDC-NHS, genipin-and TG2-treated films. All films are well-adhered to the well-plates although some shrinkage and loss of material is observed at the low crosslinking conditions of EDC-NHS, the non-crosslinked conditions as well as all the TG2-treated samples, around the periphery of the films as indicated by the lilac lines.