**1. Introduction**

Thermoresponsive hydrogels find widespread applications in medicine where synthetic and protein-based hydrogels are described. The potential applications include drug delivery, tissue engineering or the separation of bio molecules [1–4]. For example, an elastin-like polypeptide sequence, attached to graphene, with a high switching rate was designed, which revealed shrinking/bending upon irradiation with NIR (near infrared) light [5]. Examples of drug delivery have been presented by various groups, such as when an elastin-like peptide (ELP) solution is loaded with an anti-tumor drug. After injection into a tumor, the ELP coacervates due to the body temperature and forms a depot from which the drug is released over some time [6]. Similarly, the loading of ELPs with bone morphogenetic protein was described to enhance mineralization [7]. In another application, ELPs were combined with chitosan to form a multilayer system, which changes its wettability state when heated above 50 ◦C [8]. This system can assist in fine tuning cellular adhesion.

The temperature-induced contraction has an identical root for synthetic as well as protein-based polymers. All these polymers exhibit a lower critical solution temperature (LCST) upon which they become insoluble. The reason for this behavior is the imbalance between hydrophilic–hydrophobic interactions between a polymer and a solvent. Hydrophobic segments along a polymer chain can reduce their solvent-accessible surface area upon an increased temperature by aggregation, which exerts a pulling force on the non-contracting network segments. In fact, the driving force for the contraction is the

**Citation:** Wilharm, N.; Fischer, T.; Hayn, A.; Mayr, S.G. Structural Breakdown of Collagen Type I Elastin Blend Polymerization. *Polymers* **2022**, *14*, 4434. https://doi.org/10.3390/ polym14204434

Academic Editors: Ariana Hudita and Bianca Galˇ a¸ˇteanu

Received: 20 September 2022 Accepted: 18 October 2022 Published: 20 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

entropy gain for the solvent molecules. Water molecules around hydrophobic segments are highly ordered but with an increasing temperature this order is disrupted and the hydrophobic segments can associate and fold. The contraction is then actually induced when the entropy gain by the released water molecules is greater than the enthalpy gain by water binding to the polymer [9]. Poly(N-isopropylacrylamide) (PNIPAM) is one of the most investigated thermoresponsive polymers as its transition temperature is relatively insensitive to environmental conditions and is in the physiological regime (~32 ◦C) [10]. However, PNIPAM polymers have been shown to reduce cell viability for different polymerization types as well as different cell types [11]. Elastin is, therefore, a prime candidate for bio compatible thermoresponsive hydrogels as it is composed of alternating hydrophilic and hydrophobic segments and has already been shown to exhibit a LCST [12]. Recently, a collagen elastin thermoactuator with a tunable transition temperature in the physiological temperature regime was designed [13]. It was demonstrated that the incorporation of elastin from bovine neck ligament into a 2 mg/mL type I collagen gel resulted in a reversible thermoswitchable system with a transition temperature in the physiological temperature regime. As the system showed a temperature-induced phase transition like a volume contraction, it was argued that this process can be described by Euler buckling, which refers to the buckling of a rod under an axial critical load. In fact, two cases are possible when collagen and elastin are polymerized: the formation of individual fibers between collagen fibers ("perpendicular") and the incorporation of elastin monomers in a parallel manner ("lateral") into a collagen fiber. Although a lateral fiber alignment seemed likely as the buckling behavior was observed, convincing experiments have been lacking so far. We now present insights into the polymerization features of an elastin collagen hydrogel with significant evidence that elastin is laterally incorporated into the collagen fiber. Both elastin and collagen are the main components of connective tissue and exhibit distinct features. While elastin is structurally heterogeneous as the hydrophilic segments contain some *α*-helix and the hydrophobic segments are mostly of a random coil design, collagen is relatively homogeneous as three single peptide chains form a collagen triple helix [12,14]. The elastin is expressed in the endoplasmic reticulum, transported outside of the cell and then bound to micro fibrils, where, by a complex mechanism involving the crosslinking of lysine residues, an elastin fiber is formed with elastin on the inside and several micro fibrillar proteins on the outside [15]. Collagen fibrils are formed by an association of collagen triple helical monomers via an enzymatic crosslinking of lysine residues, and several fibrils then associate into fibers; however, our preparation steps were devoid of any crosslinking steps, so that the interaction between the collagen and elastin were dominated by an electrostatic interaction [16].

This work aims to present evidence that the structural metrics pore size and fiber diameter of a type I collagen hydrogel are not significantly changed upon the addition of bovine elastin. As we have found evidence supporting our thesis, we conclude that elastin monomers attach in a parallel fashion to the collagen fibers. This agrees with our own earlier studies with circular dichroism experiments, where we saw a systematic decrease in the helical structures in collagen and elastin after polymerization [13].

#### **2. Experiments**

#### *2.1. Hydrogel Preparation*

Collagen hydrogels for all experiments in this study were prepared using the same protocol as described before [13]. The basis for all the hydrogels was a mixture of collagen I monomers from rat tail (collagen R, 0.4% solution and Cat. No. 47256.01; SERVA Electrophoresis, Heidelberg, Germany) and bovine skin (collagen G, 0.4% solution and Cat. No. L 7213; Biochrom, Berlin, Germany) in a mass fraction of 1:2, respectively. To initiate the polymerization of the monomer mixture solution, a 1 M phosphate buffered solution containing disodium hydrogen phosphate (Cat. No. 71636, Merck KGaA, Darmstadt, Germany), sodium dihydrogen phosphate (Cat. No. 71507, Merck KGaA, Darmstadt, Germany) and ultrapure water was added to produce a final pH value of 7.5, ionic strength

of 0.7, and a phosphate concentration of 400 mM. To produce the collagen–elastin hydrogels, appropriate amounts of elastin powder (elastin, Cat. No. 6527, Merck KGaA, Darmstadt, Germany) were added to the buffer solution prior to the polymerization. All solutions were kept on ice. The polymerization of the final solutions was initialized by placing the samples in an incubator at 37 ◦C.

#### *2.2. UV/VIS Plate Reader Experiments*

The experiments were performed on a TECAN infinite® 200 plate reader (absorbance mode, 405 nm, target temperature 37 ◦C, 25 flashes, and a sampling rate of 1/min; TECAN Trading AG, Männedorf, Switzerland) using flat bottom 96-well plates (Carl-Roth GmbH, Karlsruhe, Germany) for the sample preparation and measurement. For a single experiment, three solutions were prepared directly before the measurement, namely, (I) 1.2 mL of a 2 mg/mL collagen solution (0.4 mL R, 0.8 mL G, each 4 mg/mL of stock solution), (II) 1.2 mL of a 2 mg/mL collagen solution (same as above) with 0.6 mg of elastin and (III) 1.2 mL of a 2 mg/mL collagen solution (same as above) with 1.2 mg of elastin. A 200 μL amount of the final solution was filled in each well, resulting in 6 wells of a 96-well plate per condition and 18 wells for all three conditions. The remaining 78 wells were filled with distilled water to ensure high humidity during the polymerization and to counteract the dehydration of the samples.
