Fish Collagen Promotes the Expression of Genes Related to Osteoblastic Activity

Tilapia type I atelocollagen (TAC) is a strong candidate for clinical application as its biological scaffold due to a high degeneration temperature and biologically safe properties. The aim of this study was to confirm the biological effects of TAC in vitro on osteoblastic cells, simulating its clinical application.The proliferation and differentiation of typical preosteoblasts, MC3T3-E1 cells, were investigated using a microarray analysis, staining assay for mineralization, and real-time PCR analysis of the expression of mineralization-related genes. The mRNA expression of 10 genes involved in proliferation and differentiation increased after 3-day culture on an TAC gel, with an average balanced score ratio exceeding 1.5 compared to the control. After two weeks of culture, all three experimental groups showed stronger alkaline phosphatase staining than after one week. The genes expression of alkaline phosphatase, osteocalcin, and bone sialoprotein increased under the experimental conditions. The gene expression of osteopontin did not increase, and no statistical differences were noted among the three experimental groups.The present and previous findings suggest that TAC is not only a suitable alternative to collagen products originating frommammals but also a novel biomaterial with cell differentiation ability for regenerative medicine.


Introduction
The research and development of nonmammalian natural biomaterials will improve the safety of scaffold and carrier in regenerative medicine, which are meaningful and beneficial, due to the global prevalence of dangerous infectious diseases (zoonosis) such as bovine spongiform encephalopathy, avian and swine influenza, tooth-and-mouth disease in cows, pigs, and buffalos, Ebola hemorrhagic fever, and Zika fever [1]. Furthermore, collagen generated from porcine and bovine sources cannot be used due to religious objections in some countries. Fish, which are the most genetically distant relatives from mammals, have recently attracted attention as an alternative source of collagen. Fish collagen (FC) derived from the scales, skin, and bone has interested many laboratories worldwide for its bioactive properties, such as excellent biocompatibility, low antigenicity, high biodegradability, and cell proliferation potential [2,3].
The weakest point of fish collagen is its relatively low denaturation temperature ( ), which indicates a poor stability in clinical applications. However, our recent experiment using calorimetry revealed that of collagen solution derived from the tropical fish, tilapia (tilapia type I atelocollagen [TAC]), was 35-36 ∘ C [4], indicating that the abovementioned weak point has been completely overcome. In addition, the biological safety of TAC has been confirmed using various test methods recommended with ISO standards [5]. The long-term storage of TCA under frozen conditions for over 1 year has already been demonstrated in our laboratory (unpublished data). These data and findings strongly support TAC as a candidate for clinical application as a biological scaffold.
In the present experiment, we confirmed the biological effects of TAC in vitro on osteoblastic cells simulating its clinical application as a suitable alternative to collagen products 2 International Journal of Polymer Science originating from mammals. The proliferation and differentiation of MC3T3-E1 cells, which are typical preosteoblasts, were investigated using a microarray analysis, staining assay for mineralization, and real-time PCR analysis (RT-PCR) to determine the expression of calcification-related genes.

Total RNA Isolation for Microarray.
For FC gel group, 1 mL of 0.1% TAC was added to a 60 mm culture dish. TCA solution was gelled at 37 ∘ C for 30 min in a condition of 5% CO 2 and air. MC3T3-E1 cells were seeded in a 60 mm culture dish covered with or without TAC gel at a density of 1.5 × 10 6 cells. After 3-day culture, total RNA from cells was isolated using TRIzol reagent (1 mL/10 cm 2 ) (Invitrogen, Carlsbad, CA, USA) and purified in accordance with the manufacturer's instructions [6]. [6]. The cRNA was amplified and hybridized to an Affinity Mouse Genome 430 2.0 array (for 39,000 transcriptional products), scanned by an Affymetrix scanner, and calculated using the Affymetrix Expression Console6. [6]. We then established the criteria for upregulated genes ( score > 3.0, ratio > 1.5fold).

RT-PCR following Microarray.
The MC3T3-E1 cells were seeded and cultured ( = 3 for FC group, = 3 for control group) under the same conditions as in the experiment for total RNA isolation for microarray. The primers are indicated in Table 1. The analyses of RT-PCR were carried out according to the similar methods to our recent study [6], except the following two steps. The first was that the protocol of amplification and quantification was 40 cycles (58-59 ∘ C, 1 min). The second was that, in the final calculations, the data were normalized by ribosomal protein S17.

Alkaline Phosphatase (ALP) Staining.
For the FC gel group, 90 L of 0.1% TAC was poured into each well. TCA solution was gelled at 37 ∘ C for 30 min in an atmosphere of

RT-PCR for Calcification-Related Genes.
For the FC gel group, 1 mL of 0.1% TAC was added to a 60 mm culture dish. TCA solution was gelled at 37 ∘ C for 30 min in a condition of 5% CO 2 and air. MC3T3-E1 cells were seeded in a 60 mm culture dish covered with or without TAC gel at a density of 1.5 × 10 6 cells. The abovementioned four groups were also prepared for this experiment ( = 3 for each group). After three days of culture, RT-PCR was applied to check the expression of four calcification-related genes: ALP, osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). All the methods were similar to the abovementioned methods. The primer designs for these genes are represented in Table 2. 2.9. Statistical Analyses. All data were represented as the mean ± standard deviation (SD). The statistical significance ( < 0.05) of the differences between the two different experimental groups was demonstrated using a paired Student'stest.

Genes Expression to Proliferation and Differentiation.
Microanalysis revealed 72 of the upregulated genes ( score > 3.0). The following 10 genes related to proliferation and differentiation of osteoblastic cells were compared with the control using the RT-PCR: vitamin D receptor (Vdr), lumican (Lum), matrix metallopeptidase 13 (Mmp13), SMAD family   Table 3). The RT-PCR analyses revealed that the mRNA expression of these 10 genes also increased after 3day culture on FC gel, with an average balanced score ratio exceeding 1.5 compared to the control (Figure 1). (Figure 2). In group (1) after 2 weeks of culture, very-weak-intensity ALP staining was observed, similar to that in group (2) after 1 week. After 2 weeks of culture, each group showed stronger staining than after 1 week. On the whole, the spindle-like shape was observed in groups (3) and (4) (Figures 2(c), 2(g), and 2(d), 2(h)). The intensity of staining increased with increasing group number from group (2) to group (4).

Gene Expression for Calcification.
The RT-PCR analyses demonstrated that the mRNA expression of four calcification-related genes in three experimental groups was analyzed after 3-day culture, compared to the control group (1) (Figure 3). Although the gene expression of ALP, OCN, and BSP increased with increasing group number from group (2) to group (4), a significant increase was recognized in BSP gene expression ( < 0.01). The raw data of OPN expression of OPN was around 1.0 in three groups, although there was a significant increase between group (2) and group (3) ( < 0.05).

Discussion
Cell culture method using the type I collagen gel was first reported by Elsdale and Bard [7]. The cells have well grown within a three-dimensional (3D) structure, as the conditions more closely resemble those of the in vivo condition of the osteoblast than the traditional two-dimensional (2D) cultures. Cells growth in 3D gels showed positive mineral staining and induction of osteoblastic marker genes earlier than cell growth in 2D cultures [8]. Although the present culture conditions were not the exact 3D gel culture system, the present environment was remarkably similar to a 3D culture environment, as a low-concentration TCA solution was gelled to mimic wet and elastic conditions [9]. Microarray and RT-PCR analyses for checking the genes involved in proliferation and differentiation in MC3T3-E1 cells demonstrated that the score data using the standard deviations presented the effect of FC on MC3T3-E1 cells more clearly than the original ratio results. Ten genes, including Wif1, a secreted modulator of Wnt proteins [10], increased expressions compared with a control condition. This datum is the first to reveal that FC directly accelerates the activity of MC3T3-E1 cells in order to maintain the preosteoblast nature.
In the present experiment, the function of MC3T3-E1 cells was accelerated after three-day culture in FC-positive or BGP-positive group, which was confirmed by the increase of the expression of calcification-related genes, except OCN. ALP is an early expression marker essential for osteoblast proliferation [11], which was well demonstrated by the present ALP staining findings and the typical change in the cell shape. Furthermore, the time lapse between mRNA expression (3 days) and protein synthesis (7 days) seemed reasonable. Although OPN, OCN, and BSP are generally used as differentiation markers for osteoblasts [11][12][13], OPN was also reported to act as a mineralization inhibitor to regulate crystal growth [14,15]. The present PCR data of OPN clearly demonstrated this osteoclastic function of OPN.
Although the intracellular signal transduction pathway and the related gene expression in osteoblast differentiation have been widely investigated [16][17][18][19][20][21][22], the direct trigger mechanisms for osteoblast proliferation and differentiation by collagen have not yet been clarified. Recently, an interesting study revealed that adhesion of human mesenchymal stem cells on tilapia collagen in vitro was faster and higher than that on porcine collagen or a noncoated surface, probably due to the texture flexibility and surface softness of the tilapia scale collagen, making the binding between collagen and integrin easier to trigger and stimulate both proliferation and differentiation of preosteoblasts [23]. A 0.1% TAC solution was used to make a gel in the present FC group. The nature of this gel is very soft, even after 2 weeks in the CO 2 incubator, and is thought to be possessed of sufficient flexibility and surface softness. This means that cultured cells may also behave as they would in a 3D culture system.
Proper biodegradation and suitable biocompatibility are indispensable for biomaterials. The biodegradation of collagen is closely linked to and dependent on the constitution of amino acids in a given species. Compared to bovine collagen, the contents of methionine, isoleucine, leucine, hydroxylysine, and lysine are lower in TAC [24]. As these amino acids are related to the collagen stiffness, the tilapia collagen can be easily degraded in vivo. The characteristics of these amino acids give TAC its abovementioned nature.
From our previous data [4,5] and the present results, TAC may represent not only a promising alternative to mammalian and avian collagen products but also a novel biomaterial with cell differentiation ability. The final goal with TAC is its clinical application as a scaffold in regenerative medicine. The enzymatic digestion time for atelotreatment and the elimination of enzyme in TAC are controlled to a clinical level (personal communication). The preliminary experimental data concerning pulp regeneration [24] using TAC as a scaffold revealed no rejection reaction on the newly regenerated pulp tissue in dogs (unpublished data). These findings suggest that TAC may be useful for clinical application after further animal experiments.

Conclusion
The present in vitro experiments using osteoblasts clearly revealed that TAC is not only a suitable alternative to collagen products originating from mammals for cell proliferation and differentiation as the scaffold for regenerative medicine.