**1. Introduction**

Hydrogels are well suited as scaffolds for tissue engineering due to their characteristics resembling natural extracellular matrices. Hydrogels can be applied in various biomedical fields, such as injectable hydrogels or printable bioinks for space-filling or cell/biological factor delivery [1]. Silk fibroin is a naturally derived fibrous protein which is widely used as a base material in hydrogel fabrication, due to its self-assembly, mechanical stability of the gels, and biocompatibility [2]. In this work, silk fibroin derived from two different sources, *Bombyx mori* silk cocoons and recombinant spider dragline silk proteins, were chosen to form blended hydrogels, and their cytocompatibility was tested in vitro.

Combining two materials is an approach to gain the advantage from both materials as well as to overcome some limitations to achieve products with desired features [3]. Silk fibroin (SF) can be derived at high amounts from silkworms by isolating the SF solution from silk glands or dissolving silk cocoons with the drawback of some inhomogeneities common

**Citation:** Laomeephol, C.; Vasuratna, A.; Ratanavaraporn, J.; Kanokpanont, S.; Luckanagul, J.A.; Humenik, M.; Scheibel, T.; Damrongsakkul, S. Impacts of Blended *Bombyx mori* Silk Fibroin and Recombinant Spider Silk Fibroin Hydrogels on Cell Growth. *Polymers* **2021**, *13*, 4182. https:// doi.org/10.3390/polym13234182

Academic Editors: Ariana Hudita and Bianca Gălă¸teanu

Received: 27 October 2021 Accepted: 25 November 2021 Published: 29 November 2021

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**Copyright:** © 2021 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/).

to all nature-derived materials. However, SF solution can be produced under certified conditions, and SF is already available from several companies, such as Fibrothelium GmbH, Aachen, Germany, Sigma-Aldrich, MO, USA, and Advanced Biomatrix, CA, USA. Glycinealanine repeats of SF can form beta sheet structures, which is relevant for self-assembly as well as the physical strength of the obtained materials [4]. However, self-gelation of SF is extremely slow (ca. 7–12 days depending on the SF concentration) [5], which is impractical in various applications, especially for cell encapsulation. Several strategies have been applied to accelerate the self-assembly process of SF, including an application of physical or mechanical forces, an addition of chemicals, as well as a simple blending with other polymers. Regarding the works from Mandal BB's group, non-mulberry SF, with its primary structure containing a high ratio of alanine-glycine and poly-alanine sequences, was simply blended with *B. mori* SF, and rapid gelation can be achieved. The biological properties were drastically improved due to the presence of arginine-glycine-aspartic acid (RGD) motifs in non-mulberry SF [6–9].

The recombinant spider silk protein (eADF4(C16)), derived from ADF4 of the dragline silk protein of *Araneus diadematus* containing poly-alanine sequence, can be spontaneously assembled into hydrogels within hours depending on the protein concentration or the ionic strength of the solution [10,11]. The advantage of this recombinant protein is its large availability with continuous properties. eADF4(C16) is a commercially available spider silk protein from AMSilk GmbH, Martinsried, Germany. Major limitation of eADF4(C16) is cell adhesion and proliferation unless the protein is genetically modified, e.g., with a tag comprising the cell adhesion motif RGD [12]. Hence, binary blending of SF and eADF4(C16) could expectedly be beneficial in enhancing the interaction with cells, as well as accelerating the gelation process within a range suitable for practical uses.

Herein, the gelation of SF and eADF4(C16) blends was evaluated. Since the hydrogels were proposed to serve as cell-loaded substrates, the interaction with primary cells and cell lines was tested. Physico-chemical properties of the hydrogels, which could affect the cellular behavior, namely micromorphology, hydrophobicity, and protein diffusivity, were also identified. This work as a proof-of-concept study provides information of blended SF:eADF4(C16) hydrogels for further applications e.g., cell-encapsulation for cell delivery or injectable or printable materials.
