Effects of phosphate-buffered saline concentration and incubation time on the mechanical and structural properties of electrochemically aligned collagen threads

A key step during the synthesis of collagen constructs is the incubation of monomeric collagen in phosphate buffer saline (PBS) to promote fibrillogenesis in the collagen network. Optimal PBS-treatment conditions for monomeric collagen solutions to induce gelation are well established in the literature. Recently, a report in the literature (Cheng et al 2008 Biomaterials 29 3278-88) showed a novel method to fabricate highly oriented electrochemically aligned collagen (ELAC) threads which have orders of magnitude greater packing density than collagen gels. The optimal PBS-treatment conditions for induction of D-banding pattern in such a dense and anisotropic collagen network are unknown. This study aimed to optimize PBS treatment of ELAC threads by investigating the effect of phosphate ion concentration (0.5×, 1×, 5× and 10×) and incubation time (3, 12 and 96 h) on the mechanical strength and ultrastructural organization by monotonic mechanical testing, small angle x-ray scattering and transmission electron microscopy (TEM). ELAC threads incubated in water (no PBS) served as the control. ELAC threads incubated in 1× PBS showed significantly higher extensibility compared to those in 0.5× or 10× PBS along with the presence of D-banded patterns with a periodicity of 63.83 nm. Incubation of ELAC threads in 1× PBS for 96 h resulted in significantly higher ultimate stress compared to 3 or 12 h. However, these threads lacked the D-banding pattern. TEM observations showed no significant differences in the microfibril diameter distribution of ELAC threads treated with or without PBS. This indicates that microfibrils lacked D-banding following electrochemical alignment and the subsequent PBS-treatment-induced D-banding by reorganization within microfibrils. It was concluded that incubation of aligned collagen in 1× PBS for 12 h results in mechanically competent, D-banded ELAC threads which can be used for the regeneration of load bearing tissues such as tendons and ligaments.