"Statement of Purpose: The development of a hydrogel that could be injected and cured in vivo has gained increasing attention. Collagen has been widely investigated as a thermogel in which there are a lot of ionic interaction, hydrophobic interaction, and hydrogen bonding; however, it must be chemically crosslinked also. Diels-Alder cycloaddition occur under mild conditions without the need for any catalyst, toxic solvent or external activation like UV-irradiation which makes it a pragmatic choice for biomedical application and in particular for developing in situ crosslinked injectable gels. Hence, taking into account the kinetic characteristics of the Diels–Alder reaction, hydrophobic diene-terminated collagen was synthesized and then injectability experiments were performed on fabricated gels to investigate its potential applications in minimally invasive surgery.
Methods: Diene-terminated collagen was prepared employing the nucleophilicity of the ε-amino group of the lysine and arginine side chain. Hydrogels were then fabricated by mixing modified collagen stock solution with PEG maleimide in which the final concentration of modified collagen in the gel was considered to be 2% (w/v) To evaluate the injectability/shear-thinning properties of the hydrogels, viscosity under continuous ﬂow was measured with increasing the shear rate (from 0.01 to 100 s−1), using 20 mm stainless steel parallel plate geometry on hydrogels extruded directly on the rheometer plate from a syringe. Shear-thinning experiments were performed immediately, 4h and 48h after mixing all the hydrogel components. Furthermore, cardiac fetal stromal cells (CFSCs) were encapsulated in the gel network and cytocompatibility of the gels investigated with live/dead viability kit.
Results: Based on our experiments, all modified collagen gels were found to be extrudable (extrusion from a syringe) and injectable (extrusion through a 27G needle), whereas with increasing the shear rate, viscosity decreased. It should also be noted that linkage between diene and dienophile in Diels-alder reaction have dynamic nature. By increasing the temperature, the reaction rate of retro Diels-alder reaction increase, shifting the equilibrium towards the breaking of the reversible bonds. Additionally, instead of thermal energy, mechanical energy also makes the reversible polymer network becomes more dynamic, leading to retro Diels-alder reaction of cycloadducts into polymer chains. Our hypothesis is that injecting with needle act as a force-actuator, resulting in a mechanochemical coupling. From this point onward, the Diels-alder adduct should be coupled to the mechanical force which eventually trigger the retro Diels-alder reaction to release diene-terminated collagen and PEG-maleimide.
Conclusion: Given details mentioned above, the slow crosslinking of Diels-alder reaction and force-induced retro Diels-alder allows modified collagen gels to be injectable at least up to 48 hours post mixing of the gel components when stored at room temperature. Hence, this property is highly beneficial for potential clinical applications in terms of handling and administration given than the hydrogel could then be prepared prior to the surgery and then brought into the surgical room. Additionally, cell viability was higher than 80% for fabricated hydrogels for up to at least 7 days after cell encapsulation, suggesting that the engineered modified collagen gels had no cytotoxicity with CFSCs."