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Biomaterials

Biomaterials - Poster Session E

Poster O16 - Polyethylene oxide templated collagen self-assembly leads to rare D/6 collagen fibril structure

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  We have recently shown that we can produce collagen multifilament bundles comprised of thousands of monofilaments by multipin contact drawing of an entangled polymer solution consisting of collagen and poly(ethylene oxide) (PEO). The multifilament bundles are hydrated in graded concentrations of PEO and phosphate buffered saline (PBS) to promote assembly of collagen fibrils within each monofilament while preserving the structure of the multifilament bundle. Multiscale structural characterization revealed that the hydrated multifilament bundle contains properly folded collagen molecules packed in collagen fibrils containing microfibrils, staggered by exactly one-sixth of the microfibril D-band spacing to produce a periodicity of 11 nm. This D/6 structure is a rare and poorly understood polymorphic product of collagen self-assembly that was previously observed at acidic pH.
Within a collagen-PEO multifilament the collagen molecules are aligned but not self-assembled. This fact points to some kind of PEO/collagen interaction being responsible for the self-assembly of collagen into D/6 banded fibrils in PBS even so this buffer is well-known to promote self-assembly of native D-banded fibrils. In this work we are exploring treatments that could alter this PEO/collagen interaction and lead to native D-banded fibrils. We studied the effect of pH, collagen type III content, presence or absence of telopeptides, and cycling loading on the self-assembly of collagen-PEO multifilaments induced by PEO removal.

Materials and

Methods:  PEO powder (Sigma Aldrich) was dissolved in telo and atelocollagen type I (~6 mg/mL in 0.01 M HCl; Collagen Solutions) to create a PEO-collagen solution. The frozen PEO-collagen solution was then lyophilized, and the resulting lyophilized PEO-collagen sponges were dissolved in acetic acid. The final PEO-collagen solution was stored at 4°C overnight and then brought to room temperature for contact drawing. Purified type III bovine collagen (MDBioproducts) in different weight ratios to collagen type I was mixed with the PEO-collagen solution.
The bio-friendly and cost-effective contact drawing technique which has been developed in our group and uses an entangled polymer fluid to aid in monofilament formation, was used (Figure 1).
In order to examine the solution-state structure of the collagen (triple helix), far-ultraviolet spectra were collected using a Circular Dichroism spectropolarimeter (Olis DSM-20) at room temperature. The WAXS patterns were acquired using a D8 Advance X-ray diffractometer (Bruker Co.) operating at a sample-to-detector distance of 70 mm with an exposure time of 600 s. SAXS measurements were performed on a SAXSpoint 2.0 (Anton Paar) using an Eiger detector set at 576 mm from the sample. Cyclic loading was applied to the collagen multifilaments using a tensile tester (Mark-10 Corporation) equipped with 10 N force sensor, at a crosshead speed of 6 mm/min.

Results, Conclusions, and Discussions: It is important to assess whether collagen type I is in its native triple-helical structure before making collagen fibers. In Figure 2A, a positive band was observed at around 221 nm, which is characteristic of collagen triple helices.
The molecular alignments of the collagen, including the intermolecular lateral packing of collagen molecules and the helical rise per residue distance, could be observed using WAXS after washing the fibers treated with different treatments. The patterns were almost identical to those of native collagen (Figure 2B).
Supramolecular structure of collagen molecules within multifilament was analyzed by SAXS (Figure 2C). The PEO-collagen multifilament did not show a periodic SAXS pattern, indicating that collagen molecules do not self-assemble during the fiber pulling process. However, after washing the PEO-collagen multifilament, several arcs appeared. These arcs can be indexed as the 6th, 7th, 8th, and 9th orders of a D-band repeat.
To our surprise, we found that the D/6 structure is insensitive to most of the attempted treatments pointing to the robustness of the PEO/collagen interaction and the complexity of the collagen self-assembly landscape. Since the stagger of the collagen molecules is related to the sequence of the side groups on the surface of the α-chains, most of the treatments were not able to effectively alter this sequence not even doping with collagen III. We also expected to see a better-defined supramolecular structure in the fibrils formed using telocollagen, as the native structure of collagen is preserved, compared to fibrils formed using atelocollagen, which has cleaved telopeptides. Interestingly, there was no significant difference between them. In some conditions, we observed even the opposite in the atelocollagen fibrils.

Given that the nanoscale mechanics of the fibrils are related to D-periodicity and that cells might be sensitive to nanoscale topography of collagen fibers, studying the aggregation of collagen fibrils with different structures is of great interest. Although most of the treatments in this study did not alter the robust D/6 polymorph of our collagen fiber, further investigations are ongoing. These include examining the effects of ionic strength, using different polymers, and blending collagen with other biopolymers like heparin.

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