**1. Introduction**

As accidents and diseases lead to tissue damage, natural tissue repair materials are a popular area of research [1]. Collagen has good biocompatibility and can promote cell proliferation and differentiation [2]. What is more, fabricated collagen nanofiber membranes have high-density pores and network structures from micro- to macro-length scales via non-thermal electrospinning. Thus, collagen electrospinning nanofiber membranes enable biomaterials to simulate the extracellular matrix (ECM) environment in vitro [3]. This, in turn, acts as a source of foundation materials for the repair of natural tissue and provides an environment for cell adhesion and proliferation. These collagen-based natural tissue repair materials are frequently used in skin, tendons, blood vessels, as well as nerve and bone regeneration [4,5]. The risk of disease transmission and religious factors have limited traditional collagen such as bovine and pig collagen [6]. Therefore, researchers have focused on searching for marine collagen. Wang et al. separated types I and V collagens from the skin of deep-sea redfish by chromatographic techniques [7]. Chen et al. separated type I collagen from the scales of the lizardfish and maintained the triple-helical structures with no cytotoxicity [8]. Recently, some Japanese scholars extracted collagen from the scales

**Citation:** Wu, S.; Yang, L.; Chen, J. Preparation and Characterization of Tilapia Collagen-Thermoplastic Polyurethane Composite Nanofiber Membranes. *Mar. Drugs* **2022**, *20*, 437.https://doi.org/10.3390/md20070437

Academic Editor: Sik Yoon

Received: 29 May 2022 Accepted: 29 June 2022 Published: 30 June 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/).

of barramundi (*Lates calcarifer*), which was found to be comparable to that of mammals and showed potential for three-dimensional cell cultivation [9]. Tilapia is a kind of freshwater and saltwater fish promoted by the FAO for aquaculture all around the world, and the production of tilapia is increasing year after year [10]. Marine tilapia collagens have also received significant attention in recent years [11–13]. However, marine collagen, without specific shape, structure, or function, cannot be directly applied. Collagen can be compounded with other materials to prepare nanofiber membranes endowed with high-density pores and a network structure with micro- to macro-lengths in scale. Researchers have developed a series of collagen-polymer nanofibrous membranes. Shue et al. fabricated a series of composite fibrous membranes incorporated with fish collagen, nanohydroxyapatite, and poly (lactic-co-glycolic acid) (PLGA) to guide bone regeneration [14]. He et al. prepared collagen-polycaprolactone (PCL) nanofiber membranes, which had diameters of at least 150 nm [15]. However, marine composite fibrous membranes often have limited mechanical properties and poor thermal stability, which hampers their use in tissue repair [16].

Thermoplastic polyurethane (TPU) is an elastomeric polymer with excellent mechanical properties, wear resistance, good elasticity, and toughness [17]. TPU also has good degradability when hydrolyzed, oxidized, or enzymatically degraded in vivo [18]. Routes to degradation of TPU can combine materials in the degradation process to retain CO2 and water and modulate changes in pH and chemical stability of the surrounding tissues [19]. Therefore, TPU promotes a stable environment for tissue regeneration; it has good mechanical properties and is environmentally friendly [20]. However, there are few reports of the micromorphology, microstructure, thermal stability, mechanical properties, and biocompatibility of collagen-thermoplastic polyurethane (Col-TPU) composite fiber membranes.

For this reason, the aim of this study was to develop collagen-based nanofibrous membranes that are suitable for the growth of fiber cells and have balanced mechanical properties, good thermal stability, and the potential to become fundamental materials for tissue repair. First, collagen was extracted from tilapia skin. Then, collagen and TPU fundamental materials were used to electrospin a series of Col-TPU nanofiber membranes. The compatibility, micromorphology, microstructure, thermal stability, mechanical properties, and biocompatibility of Col-TPU composite fiber membranes were then studied.

### **2. Results and Discussion**

### *2.1. Collagen Structure Identification*

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed three main bands, as seen in Figure 1. Tilapia skin collagen showed similar electrophoretic patterns to type I rat tail collagen consisting of two different α-chains (α1 and α2), dimeric β-chains, and γ-chains. Tilapia collagen was also consistent with rainbow trout (*Onchorhynchus mykiss*) [21] and black sea gilthead bream (*Sparus aurata*) [22]. Herein, the molecular weight of collagen was analyzed using Quantity One 4.6.0 software (Bio-Rad Laboratories, Hercules, CA, USA). Two bands had molecular weights of 130 and 121 kDa. They were assigned to two α-chains of collagen: α1 and α2 [23]. The two high-molecularweight components had weights over 200 kDa. These were identified as a β-chain consisting of two α-chains and a γ-chain consisting of three α-chains, respectively [24]. In addition, the ratio of α1 and α2 was calculated with Image J software (VERSION 1.8.0, National Institute of Mental Health, Bethesda, MD, USA); specifically, approximately 2:1 was consistent with the molecular structure of type I collagen [α1]2α2, thus indicating that the prepared TSC was type I collagen.

**Figure 1.** Preparation and characterization of collagen. (**A**) Type I rat tail collagen, (**B**) Tilapia skin collagen.

### *2.2. Structural Analysis of Col-TPU Composite Nanofiber Membranes*

### 2.2.1. Scanning Electron Microscope (SEM) Analysis

SEM (Figure 2) revealed that Col-TPU composite nanofiber membranes (Col95, Col90, Col80, Col60) are uniform and continuous with no beads and better straightness than pure TPU. They range in diameter from 112 nm to 858 nm. The porosity increased with increasing TPU ratios. The average diameters of Col100, Col95, Col90, Col80, and Col60 decreased from 379.96 ± 134.28 nm to 378.40 ± 151.87 nm, 316.80 ± 94.51 nm, 313.80 ± 102.88 nm, and 232.94 ± 87.82 nm, respectively (Table 1). The average diameters of the nanofibers decreased gradually with the increasing compound ratio of TPU. The porosity of all composite nanofiber membranes was higher than 45%, which benefits water and oxygen permeability. The appropriate porosity also facilitates cell migration and cell attachment in the resulting pore structure.

**Table 1.** SEM results of Col-TPU composite nanofiber membranes.


**Figure 2.** SEM analysis. (**A**) Col100. (**B**) Col95. (**C**) Col90. (**D**) Col80. (**E**) Col60. (**F**) TPU.
