*Article* **Evaluation of Differentiated Bone Cells Proliferation by Blue Shark Skin Collagen via Biochemical for Bone Tissue Engineering**

**Jeevithan Elango 1,†, Jung Woo Lee 1,2,†, Shujun Wang 3, Yves Henrotin 4, José Eduardo Maté Sánchez de Val 5, Joe M. Regenstein 6, Sun Young Lim 2, Bin Bao 1,\* and Wenhui Wu 1,3,7,\***


Received: 22 August 2018; Accepted: 17 September 2018; Published: 25 September 2018

**Abstract:** Collagen from a marine resource is believed to have more potential activity in bone tissue engineering and their bioactivity depends on biochemical and structural properties. Considering the above concept, pepsin soluble collagen (PSC) and acid soluble collagen (ASC) from blue shark (*Prionace glauca*) skin were extracted and its biochemical and osteogenic properties were investigated. The hydroxyproline content was higher in PSC than ASC and the purified collagens contained three distinct bands α1, α2, and β dimer. The purity of collagen was confirmed by the RP-HPLC profile and the thermogravimetric data showed a two-step thermal degradation pattern. ASC had a sharp decline in viscosity at 20–30 ◦C. Scanning electron microscope (SEM) images revealed the fibrillar network structure of collagens. Proliferation rates of the differentiated mouse bone marrow-mesenchymal stem (dMBMS) and differentiated osteoblastic (dMC3T3E1) cells were increased in collagen treated groups rather than the controls and the effect was dose-dependent, which was further supported by higher osteogenic protein and mRNA expression in collagen treated bone cells. Among two collagens, PSC had significantly increased dMBMS cell proliferation and this was materialized through increasing RUNX2 and collagen-I expression in bone cells. Accordingly, the collagens from blue shark skin with excellent biochemical and osteogenic properties could be a suitable biomaterial for therapeutic application.

**Keywords:** blue shark collagen; osteogenic activity; Runx2; differentiated mesenchymal stem cell; osteoblast; proliferation

#### **1. Introduction**

There are three major types of protein present in fish, such as sarcoplasmic (25–30%), myofibrillar (66–77%), and stroma protein (1–5%). Among them, stromal proteins are mostly located in the interstitial space of muscle cells and the extracellular membrane. One of the most available stromal proteins in fish is collagen. It is extracted from different part of fish such as bones, scales, skin, swim bladder, and fins. Currently, collagen from the marine organism has been widely investigated due to its excellent biological activity with low or no side effect as an alternative to mammalian collagen [1,2]. Collagen is graded based on its biochemical, functional, and rheological characteristics, which may depend on fish species and the manufacturing method. Based on the quality and bioactivity, fish collagen can be used as a drug in the biomedical industries or as a food supplement in food industries.

In tissue engineering, fish collagen is used in the form of films, scaffolds, sponge, hydrogel, microspheres, and composite biopolymers. Wang et al. [3] studied the hemostatic properties of type I collagen to treat tissue burns. In another study, type I collagen 3D matrix was used as a potential biomaterial for heart regeneration [4]. It is well known that the growth and characteristic of cells such as proliferation, adhesion, and maturation were enhanced by collagen labeling [5]. It was found that the inflammatory response of tilapia collagen biomaterials in rabbits was similar to that of collagen from porcine or polyethylene [6]. In addition, the collagen from jellyfish could induce an immune response similar to that of bovine collagen [7]. Sugahara et al. [8] reported that jellyfish collagen produced by pepsin digestion up-regulated the production of immunoglobulin IgM in the human hybridoma cells and IgM and IgG in human peripheral blood lymphocytes due to the presence of telopeptides. Our recent research confirmed the biocompatible and non-immunogenic effect of acid soluble- and pepsin soluble-collagen isolated from shark cartilages and tilapia skin and these collagens did not elicit immune response in in-vivo and in-vitro models [9,10]. In that sense, collagen from shark can be used as a potential biomaterial for bone tissue engineering.

Indeed, shark collagen has been widely used in bone tissue engineering application due to its excellent biocompatible, osteoconductive, osteoinductive, and natural bone biomimetic properties. The collagen material used in bone tissue engineering not only regulates morphological properties but also maintains appropriate cues for regulating cellular processes during bone formation. It was reported that the collagen-based biomaterial implanted in rabbit induced neotendon and neoligament formation [11,12]. Our previous study revealed the osteogenic potential of whale shark bone collagen on mesenchymal stem cells and primary osteocytes [13]. In another study, composite scaffold 3D matrices were prepared by mixing acid soluble collagen from blue shark (*Prionace glauca*) skin with calcium phosphates from the teeth of *Prionace glauca* and *Isurus oxyrinchus* and tested the effect of a composite collagen scaffold 3D matrix in the proliferation of osteoblast-like cells, Saos-2 [14]. However, the osteogenic properties of pure collagen isolated from blue shark skin need to be addressed before being used as a biomaterial in bone tissue engineering, since the osteogenic potential of fish collagen is profoundly influenced by the molecular composition and arrangement, which is thought to be varied by different extraction methods. Considering the above hypothesis, for the first time, we explored the osteogenic response of type I collagens (Acid and Pepsin soluble) isolated from blue shark (*Prionace glauca*) skin using differentiated mouse bone marrow-mesenchymal stem (dMBMS) and differentiated osteoblastic (dMC3T3E1) cells and their relationship with the biochemical and functional properties of collagen in the present study.
