Photochemical cross-linking for collagen-based scaffolds: a study on optical properties, mechanical properties, stability, and hematocompatibility.

Collagen presents an attractive biomaterial for tissue engineering because of its excellent biocompatibility and negligible immunogenicity. However, some intrinsic features related to the mechanical stability and thrombogenicity limit its applications in orthopedic and vascular tissue engineering. Photochemical cross-linking is an emerging technique able to stabilize tissue grafts and improve the physicochemical properties of collagen-based structures. However, other important properties of collagen-based structures and the effect of processing parameters on these properties have not been explored. In this study, we aim to investigate the dose dependence of tensile and swelling properties on two parameters, namely, laser energy fluence and rose Bengal photosensitizer concentration. We also study the compression properties using cyclic compression test, long-term stability using subcutaneous implantation, and hematocompatibility using platelets adhesion test, of cross-linked collagen structures. Moreover, because limited optical penetration in turbid media is the major obstacle for light-based techniques, we also characterize the optical properties, which partially determine the effective optical penetration depth in collagen gel samples, during photochemical cross-linking. Laser energy fluence and rose Bengal concentration are important parameters affecting the cross-linking efficiency, which was characterized as the mechanical and the swelling properties, in a dose-dependent manner. Under the experimental conditions in this study, the peak fluence was 12.5 J/cm2 and the minimal rose Bengal concentration for effective cross-linking was >0.00008% (0.786 micromol). Photochemical cross-linking also enhanced the compression strength and long-term stability of collagen structures without compromising the tissue compatibility. Furthermore, photochemical cross-linking reduced platelet adhesion and abolished fibrin mesh formation, thereby improving the hematocompatibility of collagen structures. These results suggest the feasibility of using the photochemically cross-linked collagen structures for orthopedic and vascular tissue engineering. Finally, the effective optical penetration depth in collagen gel samples is wavelength and rose Bengal concentration dependent, and was approximately 12 mm at 514 nm at 0.001% (9.825 micromol), the rose Bengal concentration mostly used in this study.

Effects of photochemical crosslinking on the microstructure of collagen and a feasibility study on controlled protein release.

Photochemical crosslinking is an emerging technique able to modify the physicochemical properties of collagen. However, whether this technique can be used to modify collagen-based structures for drug delivery has not been studied. This study demonstrated that the microporous structure of photochemically crosslinked collagen was affected by rose Bengal and laser energy level. Using the optimized process parameters, the authors fabricated photochemically crosslinked collagen structures encapsulated with sample proteins and demonstrated that photochemical crosslinking reduced the initial burst effect and protein release without compromising the protein bioactivity. The fiber meshwork in collagen structures was also characterized, and it was found that photochemical crosslinking did not significantly alter the mesh size. This study reports the effects of photochemical crosslinking on the microstructure of collagen structures and suggests the feasibility of using photochemically crosslinked collagen structures for controlled protein release.

Fabrication of nano-fibrous collagen microspheres for protein delivery and effects of photochemical crosslinking on release kinetics.

Protein compatibility is important for protein drug delivery using microsphere-based devices. Collagen has excellent protein compatibility but has poor mechanical stability for microsphere fabrication and open meshwork for controlled release. In this study, a protein-compatible fabrication method for injectable collagen microspheres has been developed. The surface morphology, interior microstructure and protein release characteristics of collagen microspheres were investigated. Moreover, effects of photochemical crosslinking on these characteristics were also studied. Finally, the mechanisms governing the protein release and the retention of protein bioactivity were studied. Stable and injectable collagen microspheres consisting of nano-fibrous meshwork were successfully fabricated under ambient conditions in an organic solvent and crosslinking reagent-free manner. These microspheres have open meshwork and showed large initial burst and rapid release of proteins. Photochemical crosslinking significantly reduced the initial burst effect and controlled the protein release in a photosensitizer dose-dependent manner without significantly altering the mesh size. We further demonstrated that there was significantly higher protein retention within the photochemically crosslinked collagen microspheres as compared with the uncrosslinked, suggesting a secondary retention mechanism. Lastly, both surfactant treatment and photochemical crosslinking did not compromise the bioactivity of the encapsulated proteins. In summary, this study reports a novel collagen microsphere-based protein delivery system and demonstrates the possibility to use photochemical crosslinking as the secondary retention mechanism for proteins.