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

Abstract

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[1] 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 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× or 10×) and incubation time (3, 12 or 96 hours) on the mechanical strength and ultrastructural organization by monotonic mechanical testing, small angle X-ray scattering and transmission electron microscopy. ELAC threads incubated in water (No PBS) served as the control. ELAC threads incubated in 1× PBS showed significantly higher extensibility compared to 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 hours resulted in significantly higher ultimate stress compared to 3 or 12 hours. However, these threads lacked D-banding pattern. TEM 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 hours results in mechanically competent, D-banded ELAC threads which can be used for the regeneration of load bearing tissues such as tendons and ligaments.

Modeling the Electromobility of Type-I Collagen Molecules in the Electrochemical Fabrication of Dense and Aligned Tissue Constructs

“…Additionally, the self-aggregation phenomena along the pI of neutrally charged collagen molecules may explain why type-I collagen threads formed using this modified isoelectric method lacked the typical d-banding pattern of natural collagenous structures. 39 Based on the neutral charge of collagen molecules along the pI, it can be inferred that the molecules will aggregate in a direction parallel the electrodes with no preferential staggered order that would contribute to the d-banding pattern. The lack of d-banding in freshly aligned fibers teased from ELAC threads has been experimentally observed by Cheng et al 9 However, treatment of aligned collagen in phosphate buffered saline at neutral pH and incubation at 37°C result in the induction of d-bands as we have shown earlier using SAXS.…”

“…The lack of d-banding in freshly aligned fibers teased from ELAC threads has been experimentally observed by Cheng et al 9 However, treatment of aligned collagen in phosphate buffered saline at neutral pH and incubation at 37°C result in the induction of d-bands as we have shown earlier using SAXS. 9,39 In vitro, the self-assembly process of type-I collagen molecules can be initiated from cold acid conditions (4°C, pH 3) to warm neutral conditions (34-37°C, pH 7.4). 41,42 This self-assembly process reveals a lag phase, with no increase in turbidity, followed by a sigmoidal growth with concomitant turbidity increase, and a final plateau when precipitation of collagen occurs.…”

“…1,9,14,21,23,39 While IEF is routinely used for protein identification and separation, its utilization for fabricating materials is novel and significant. The significance of electrochemical fabrication is in its potential to generate collagenous biomaterials whose fabric is as dense, as aligned and as strong as collagenrich tissues such as tendons and ligaments.…”

“…The significance of electrochemical fabrication is in its potential to generate collagenous biomaterials whose fabric is as dense, as aligned and as strong as collagenrich tissues such as tendons and ligaments. 9,21,39 Specifically, ELAC threads have shown mechanical properties converging to those of native tendon, 9 have shown to incorporate decorin-mimic molecules within its structure, 21 and have exhibited a periodic staggered conformation in the form of d-banding pattern. 39 Electrochemically aligned collagen threads have good in vivo biocompatibility.…”

“…9,21,39 Specifically, ELAC threads have shown mechanical properties converging to those of native tendon, 9 have shown to incorporate decorin-mimic molecules within its structure, 21 and have exhibited a periodic staggered conformation in the form of d-banding pattern. 39 Electrochemically aligned collagen threads have good in vivo biocompatibility. 22 Additionally, ELAC threads have shown tenogenic differentiation of human mesenchymal stem cells only by direct contact, without the need of growth factor supplementation.…”