**4. Discussion**

The most remarkable finding within our experiments was that the addition of elastin to a collagen solution at pH 7.5 does neither induce significant changes within the polymerization process nor structural changes within the network later. Initially, we discussed two extremes of a collagen–elastin interaction, namely, perpendicular and lateral polymerization. While we expected a mixed state between these two extremes prior to the experiments, it became quickly clear that the experiments favored the lateral state over the perpendicular and mixed state (Figure 8). This was also initially proposed as we had observed a Euler buckling-like behavior of the hybrid gels under heating in earlier experiments [13]. This phase transition-like behavior can only manifest if elastin conveys a compressive force on collagen fibers in the axial direction. In the opposite case of a perpendicular connection, one would expect a linear decline in the volume with the temperature, as the collagen network would gradually follow the contractive force conveyed by elastin. An homogenous incorporation by a lateral fiber alignment is also likely from another perspective. The persistence length lp of a polymer describes the length over which bending fluctuations are correlated, where a larger value means that the respective polymer is rather inflexible. The literature reports values for collagen of lp = 10 nm to 20 nm and for elastin of lp = 0.3 nm to 0.6 nm [37–39]; therefore, elastin monomers are around 30 times more flexible than collagen monomers. Together with only 1/4th of collagen's mass, it is easily imaginable that elastin monomers attach quickly and in a highly adaptive manner to collagen monomers.

**Figure 8.** Proposed incorporation of elastin into a collagen fibril (hydrophilic and hydrophobic segments of elastin are not shown). The random coil ends of collagen should signify the suggested interference of elastin with collagen's secondary structure during polymerization. Elastin monomers are thought to bind to collagen through local H-bonds, van-der-Waals bonds and ionic bonds, although the latter is less likely due to the low zeta potential.

## **5. Conclusions**

We showed insights into the polymerization features of elastin–collagen hydrogels. Especially, it was shown that elastin and collagen chains interact in a lateral fashion. This was directly demonstrated with the LSM recordings of collagen and collagen–elastin gels where the collagen was separately stained over the collagen–elastin and further indirectly, as the addition of elastin did not change the structural metrics pore size, fiber thickness or 2D anisotropy. Although we did not quantify changes in the axial and lateral polymerization rate, a visual inspection of the Videos S1–S4 highlights no changes in this polymerization metric after the elastin addition, i.e., the axial fiber growth still starts earlier than for lateral growth; however, the plate reader experiments revealed an elastin concentration-dependent acceleration of the polymerization rate and no signs of elastin coacervation in the presence of collagen. This is a strong sign that a lateral elastin–collagen association precedes the temperature-induced loss of the hydration shell in both, leading to homogenous elastin–collagen hybrid fibers. Further, the zeta potential experiments confirmed a similarly low potential for elastin and collagen, confirming optimal conditions for aggregation. Taken together, the addition of bovine neck ligament elastin to type I collagen solutions accelerated the polymerization rate, although no significant structural changes of the resulting gels could be observed. To generalize our findings, we showed elastin's propensity to bind to other bio polymers such as collagen in a lateral manner and our findings can help to guide the preparation of other elastin-based bio materials with or without actuatoric application.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14204434/s1, Figure S1. Left: 10 stacked images of a 2 mg/mL collagen type I gel. Right: cluster detection as indicated by black dots. These were ignored for the fiber thickness estimation as shown in Figure S2; Figure S2. Left: exemplary 2 mg/mL collagen gel. Right: fiber thickness estimation. Detection was similarly undertaken for the elastin–collagen after cluster removal as shown in Figure S1; Figure S3. Top: extended polymerization curves. Mean polymerization curves at 37 ◦C for a 2 mg/mL collagen solution as well as two collagen elastin solutions containing 20 w% (0.6 mg/mL elastin) and 33.3 w% (1.2 mg/mL elastin), respectively. Bottom: derivative of the mean of the top curves. Six wells were recorded per sample and the color-coded curves in the top figure denote one standard deviation; Figure S4. Measured zeta potential values over time. Three samples were analyzed per condition. Video S1: Collagen; Video S2: 20 Elastin + Collagen; Video S3: 33.3 Elastin + Collagen; Video S4: Comparison.

**Author Contributions:** Conceptualization, N.W.; methodology, N.W., T.F., A.H.; software, N.W., T.F., A.H.; validation, N.W., T.F.; formal analysis, N.W., T.F., A.H.; investigation, N.W., T.F., A.H.; resources, N.W., T.F., A.H.; data curation, N.W., T.F., A.H.; writing—original draft preparation, N.W.; writing—review and editing, T.F., A.H., S.G.M..; visualization, N.W., T.F.; supervision, S.G.M.; project administration, S.G.M.; funding acquisition, S.G.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was financially supported by the Deutsche Forschungsgemeinschaft (DFG–Project MA 2432/6-3) as well as the Saxonian Ministry for Higher Education, Research and the Arts (SMWK) (100331694 (MUDIPlex)) is gratefully acknowledged. The LSM employed in these studies was funded by INST 268/357-1 FUGG (project number 323490432).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We gratefully acknowledge Jan Griebel (IOM) and Nadja Schönherr (IOM) for the zeta potential measurements and discussion as well as Christian Elsner (IOM) for the plate reader and antibody experiments. This project was partially performed within the Leipzig Graduate School of Natural Sciences–Building with Molecules and Nano-objects (BuildMoNa).

**Conflicts of Interest:** The authors declare no conflict of interest.
