**1. Introduction**

Bone is a dense, complex, hierarchically structured connective tissue composed of calcified matrix, which includes 65% inorganic materials, 25% organic materials, and 10% water, cells (osteoblasts, osteoclasts, and osteocytes), and various proteins, such as osteocalcin, osteopontin, and osteoprotegerin [1,2]. Bone is one of the most important tissues in the human body and provides mechanical strength, protects vital body organs, and stores and releases minerals; also, bone is continuously subjected to various defects, injuries, and diseases [3]. Some bone-related issues cannot be self-repaired and require special treatments, such as bone grafts including autografts, allografts, and tissue-engineered bone substitutes that promote bone tissue regeneration [4]. Bioceramics including bioactive glass,

**Citation:** Kim, S.-C.; Heo, S.-Y.; Oh, G.-W.; Yi, M.; Jung, W.-K. A 3D-Printed Polycaprolactone/Marine Collagen Scaffold Reinforced with Carbonated Hydroxyapatite from Fish Bones for Bone Regeneration. *Mar. Drugs* **2022**, *20*, 344. https:// doi.org/10.3390/md20060344

Academic Editor: Sik Yoon

Received: 22 February 2022 Accepted: 23 May 2022 Published: 25 May 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/).

hydroxyapatite (HA), and calcium phosphate are promising bioactive materials that were successfully used for bone tissue regeneration applications [5].

HA (Ca10(PO4)6(OH)2) is the main component of bone and accounts for approximately 60–70% of total bone mass [6,7]. Hence, HA was reported to be a potential therapeutic material and was applied in spinal fusion surgery, bone defect treatment, bone-related surgery, and bone mass augmentation [8,9]. Although HA is a bioactive material that induces bone regeneration, this material has a lack of mechanical stability, high brittleness, and low interaction with cells. Thus, many studies are currently being conducted to composite HA with various synthetic and natural materials to improve its mechanical properties and cellular functions such as cell adhesion, migration, and differentiation [10,11].

Collagen is the main protein in the extracellular matrix (ECM), accounting for approximately one-quarter of total body protein [12,13]. Collagen is a structural protein with a triple-helical structure that is composed of repeated G-X-Y peptide units. There are 29 types of collagen, and the majority are type I, II, and III. Collagen is a particularly promising biomaterial that promotes cell adhesion, proliferation, and differentiation by providing an ECM-mimicking environment [14,15]. However, collagen isolated from land animals, such as pigs and cows, has various barriers to application in medical products, due to religious reasons and zooanthroponoses. Marine-derived collagen is a highly potent alternative to land animal-derived collagen, since there are no religious barriers or reported zooanthroponoses [16–18]. In addition, marine-derived collagen has shown a lower immune response, higher water solubility, and lower production costs than land animal-derived collagen [19,20]. Among marine organisms, *Paralichthys olivaceus* may be a good substitute for land animal-derived collagen, because of its good accessibility and availability as a byproduct in the seafood industry in the Republic of Korea [21]. However, investigations of bone regenerative scaffolds fabricated with *P. olivaceus*-derived biomaterials were rarely reported. In addition, unlike jellyfish containing a large amount of collagen type 2 and a marine sponge containing a large amount of collagen type 4, fish skins contain a large amount of collagen type 1, so it can be used as a raw material for biomaterials for tissue regeneration [22].

Polycaprolactone (PCL) is a biocompatible, biodegradable synthetic polymer that was approved by the United States Food and Drug Administration (FDA) and is widely used in the fabrication of tissue-engineered substitutes for bone regeneration applications [23,24]. Moreover, PCL has good mechanical properties such as high stiffness and strength, and slow biodegradation time (2–4 years); these properties play key roles in maintaining the appropriate mechanical strength in tissue-engineered bone substitutes fabricated with natural polymers and bioceramics [25–27].

A three-axis plotted scaffold provides a three-dimensional (3D) structural environment to facilitate osteocyte adhesion, migration, and differentiation; these scaffolds are subjects of bone regeneration research [28,29]. In particular, 3D printing is an immerging technology that can be applied to fabricate complex and personalized structures; this technique is highly reproducible, compared with other techniques such as gas foaming, hydrogel usage, and electrospinning [30]. Moreover, organic substances, such as alginate, chitosan, gelatin, and collagen, and inorganic substances, such as HA, tricalcium phosphate, and whitlockite are widely used to fabricate bone regenerative scaffolds using 3D printing technology [5,31,32]. Therefore, 3D-printed scaffolds offer benefits for patient treatment through personalization.

In this study, we fabricated a composite 3D-printed scaffold using PCL and carbonated HA (CHA), and enhanced the biological properties using a coating of marine atelocollagen (MC). MC and CHA were extracted from the skin and bones, respectively, which are byproducts of *P. olivaceus*. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) spectroscopy, and amino acid composition analyses were used to evaluate the characteristics of MC, and FTIR spectroscopy, X-ray diffraction (XRD) analysis, and energy dispersive spectroscopy (EDS) were performed to evaluate the characteristics of CHA. The fabricated CHA/MC/PCL scaffolds were then

analyzed to determine their potential for facilitating osteogenic differentiation and bone tissue regeneration through in vitro and in vivo investigations.
