*2.10. Effect of Collagen on Bone Cells*

The effect of collagens on the dMBMS and dMC3T3E1 cell growth was determined and presented in Figure 7. As shown in Figure 7, the collagens treated dMBMS and dMC3T3E1 cells showed a higher cell proliferative rate than the control cells and the rate was accelerated with increasing collagen concentration in a dose-dependent manner. Remarkably, the osteogenic effect of the collagen on the bone cell was maxima at a high concentration (50 ng/mL). Among the two cells, dMBMS cells had a higher proliferation rate in PSC treated group. Whereas dMC3T3TE1 cells showed similar cell proliferation in both ASC and PSC treated groups. A recent study by Diogo et al. [14] reported that the composite blue shark skin collagen-calcium phosphate scaffold crosslinked with 12.5% EDC/NHS accelerated Saos-2 cells metabolic activity and supported osteoblast-like cells formation, however, this study was conducted using acid soluble collagen. Conversely, in the present study, the collagen was extracted by two different methods using acetic acid and pepsin, respectively, and the proliferative effect of freeze dried collagens was tested using dMBMS and dMC3T3E1 cells.

**Figure 7.** The effect of blue shark skin collagen on differentiated bone cells proliferation. MBMS: differentiated mouse bone marrow mesenchymal stem cells, MC3T3E1- differentiated-osteoblasts, ASC-Acid soluble collagen, PSC-pepsin soluble collagen. For differentiation, MBMS and MC3T3E1 cells were cultured with osteoblast differentiation medium for 21 days. \* *p* < 0.05 vs control.

The levels of osteogenic mRNA expression of alkaline phosphatase, collagen 1 aplha1 and Runx2 were significantly increased in the collagen treated cells than the control cells, however, the osteocalcin mRNA expression was not significantly altered between the collagen and control cells (Figure 8A). Confocal laser scanning microscope showed a high number of mature bone cells in collagen treated cells compared to control cells (Figure 8B), which further substantiated the osteogenic stimulatory activities of blue shark skin collagens. Similar to the present study, type I collagen and its peptides from rat tail showed osteogenic stimulatory activities on a mesenchymal stem cell [27,28]. Recently, Chiu et al. [29] reported that the MBMS cell expressed a high the level of integrin α2β<sup>1</sup> complex upon collagen treatment. In support of the proliferation result, the levels of osteogenic mRNA of alkaline phosphatase and collagen 1 were increased in collagen treated cells. Runx mRNA expression of collagen treated cells revealed the potential osteogenic activity of collagen. These findings further justify the increased proliferation rate of collagen treated cells. Recent studies claimed that certain amino acids such as glutamine, alanine, asparagine, and glycine of collagen triggered new bone cell formation through the initiation of FAK-JNK signaling pathway via RUNX2 in MBMS cell [29,30].

**Figure 8.** mRNA (**A**) and confocal images (**B**) of blue shark collagen treated bone cells. Scale bars: 75 micrometers. MBMS: differentiated mouse bone marrow mesenchymal stem cells, MC3T3E1 differentiated osteoblasts, ASC-Acid soluble collagen, PSC-pepsin soluble collagen. For differentiation, MBMS and MC3T3E1 cells were cultured with osteoblast differentiation medium for 21 days. \* *p* < 0.05 vs. control.

The western blot analysis of the osteogenic protein expression of collagen treated cells was in agreement with the proliferation, mRNA expression, and microscopic results, which indicated the higher osteogenic protein expression of collagen I alpha I and Runx2 in collagen treated cells than in the control (Figure 9A).

**Figure 9.** Osteogenic regulatory protein expressions (Collagen I and Runx2) of bone cells (differentiated MBMS) treated with blue shark skin collagen by western blot assay. (**A**) Level of osteogenic protein expression of bone cells (**B**) Fold changes of osteogenic protein expression compared to control (GAPDH). ASC-Acid soluble collagen, PSC-Pepsin soluble collagen. For differentiation, MBMS cells were cultured with osteoblast differentiation medium for 21 days, \* *p* < 0.05.

In addition, the cells treated with PSC showed higher osteogenic regulatory protein expressions than ASC treated cells (Figure 9B). Interestingly, the Runx2 protein was highly expressed in collagen treated cells, especially in PSC treated cells. It was reported that the collagen could interact with integrin alpha1 beta 2 of the mesenchymal stem cells and trigger FAK/JNK signals through Runx2 during osteoblast differentiation [29]. In that sense, the blue shark skin collagen accrued osteoblast differentiation through upregulating the Runx2 protein expression in dMBMS cells. These findings further confirmed the admirable osteogenic properties of blue shark skin collagens.

#### **3. Materials and Methods**

## *3.1. Extraction, Purification, and Total Collagen Content of Fish Collagen*

The raw material, blue shark skin (*Prionace glauca*), was procured from a private fish processing plant, M/s. Yueqing Biological Health Care Product Co., Ltd. Zhejiang, China and chopped into small pieces. They were then homogenized in PBS prior to extraction. The pretreatment was done in order to remove water-soluble protein, non-collagen protein, and mineral content using distilled water, NaOH and ethylenediaminetetraacetic acid (EDTA), respectively. The pretreated samples were used for the extraction of acid soluble collagen (ASC) and pepsin soluble collagen (PSC) using acetic acid and acetic acid containing 1% pepsin, respectively, as per our previous method [10]. The extracted samples were purified using Sephadex G-100 column chromatography coupled with UV spectroscopy with the absorbance of 230 nm [1]. The purified samples were freeze-dried using a lyophilizer. The hydroxyproline and total collagen content of the purified samples were determined [31].
