Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen

doi:10.1016/j.jbiosc.2016.01.001

Journal of Bioscience and Bioengineering

Volume 122, Issue 2, August 2016, Pages 219-225

https://doi.org/10.1016/j.jbiosc.2016.01.001 Get rights and content

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro.

Section snippets

Collagen observation by scanning electron microscopy

Tilapia collagen was prepared using Cell Campus AQ-03A (Taki Chemical, Hyogo, Japan; 0.3%, pH 3.0). Porcine collagen were prepared with Cellmatrix I-C (Nitta Gelatin, Osaka, Japan; 0.3%, pH 3.0) for comparison. For scanning electron microscopy (SEM) observation, tilapia and porcine collagen solution were adjusted to 0.2 ml 0.1% collagen/PBS and added to a 1.5 ml tube and incubated at 30°C for 3 h. Gel-like collagen fibrils were treated with 4% paraformaldehyde (Sigma–Aldrich, SL, USA) for

Morphology of tilapia scale and porcine collagen fibrils

To compare collagen fibrils formed from tilapia scale and porcine collagen molecules, both collagen fibrils were observed by SEM. Tilapia collagen formed fibrils of 1.0–2.5 μm diameter (Fig. 1A), whereas porcine collagen formed fibrils of 0.5–1.5 μm diameter (Fig. 1B). Images also showed that tilapia collagen fibrils looked helically coiled (Fig. 1A), whereas porcine collagen showed a simpler structure with individual fibrils (Fig. 1B).

Safranin O staining of hMSCs on tilapia and porcine collagens

hMSCs were pre-cultured on tilapia and porcine

Discussion

The rapid regeneration potential of fish scales has been reported previously 8, 9, especially of tilapia scales, in which collagen is highly orientated and has the potential for rapid fibril formation (9). Compared with other fish species, the denaturing temperature of tilapia collagen is higher and suitable for tissue or cell culture at 37°C (5). Furthermore, tilapia collagen showed rapid fibril formation and facilitated early hMSC osteoblastic differentiation (3). On the other hand, the risk

Acknowledgment

We thank Dr. Hiroshi Itoh, Dainichiseika Color & Chemicals Mfg. Co., Ltd., for technical supports.

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Large and massive rotator cuff tears are challenging for surgeons because of postoperative complications such as repaired site retears. Recently, collagen extracted from fish scales has gained more attention because fish byproducts are considered a safer collagen source than other animal-derived scaffolds. This study aimed to evaluate the biological efficacy of tilapia scale–derived collagen scaffolds for rotator cuff repair in rat models.
The infraspinatus tendon was resected from the greater tuberosity of Sprague-Dawley rats. In the control group, the tendon edge was sutured directly to the humeral head. In the augmentation group, the repaired site was augmented with a tilapia scale–derived collagen scaffold. Histologic examinations were performed at 2 and 4 weeks postoperatively via safranin O and immunofluorescence staining (isolectin B4 and type II collagen) in the bone-tendon junction. For mechanical analysis, the ultimate failure load of the tendon-humeral head complex was evaluated at 6 weeks postoperatively.
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Collagen obtained from fish offal (skin, scales, and bones) is required from some religious and ethnic groups, thus indirectly increasing demands for fish collagen for biomedical applications. The limitation of fish collagen is its lower thermal stability compared to mammalian collagen. In this study, we focused on collagen extracted from scales of the marine fish barramundi (Lates calcarifer) and demonstrated the suitability for the collagen to be utilized in collagen fibril matrices (CFM). Collagen was extracted from the scales through pepsin-digestion and purified (designated as “BC”). The denaturation temperature (Td) for BC was determined to be 36.4 °C, one of the highest among fish collagens. BC formed CFM which was thermally stable at 37 °C, while Td was lower than 37 °C. This could be explained by the fast fibril formation, initiating at temperatures near 20 °C in a temperature-elevated process. As a result, the NIH3T3 cells were successfully encapsulated in the CFM of BC and cultured three-dimensionally for 7 d. The cells spread and exhibited well-developed pseudopodia in the CFM of BC as observed in the CFM of pig collagen matrices. This is the first report on fish CFM used for conventional 3-D cell culture.
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Citation Excerpt :
Collagen isolated from its skin is easily obtained and has shown similar features to that of mammals [18,19]. Also, early studies demonstrated that the high orientation of marine collagen can be advantageous to guide cartilage regeneration [20,21]. In line with this, this work addressed the potential of blue shark Prionace glauca (PG) skin collagen-based 3D structures to promote human adipose stem cells (hASC) differentiation into a chondrogenic lineage with and without exogeneous stimulation.
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^§: Present address: Department of Precision Science and Technology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

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Journal of Bioscience and Bioengineering

Section snippets

Collagen observation by scanning electron microscopy

Morphology of tilapia scale and porcine collagen fibrils

Safranin O staining of hMSCs on tilapia and porcine collagens

Discussion

Acknowledgment

References (24)

Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major

J. Struct. Biol.

Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas

Int. J. Biol. Macromol.

Osteoblastic activity and estrogenic response in the regenerating scale of goldfish, a good model of osteogenesis

Life Sci.

Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion

Blood

Fish bone chemistry and ultrastructure: implications for taphonomy and stable isotope analysis

J. Archaeol. Sci.

Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins

Food Hydrocoll.

Serum enhancement of human endothelial cell attachment to and spreading on collagens I and IV does not require serum fibronectin or vitronectin

J. Cell Sci.

Construction and characterization of a tissue-engineered oral mucosa equivalent based on a chitosan-fish scale collagen composite

J. Biomed. Mater Res. B Appl. Biomater.

Rapid oriented fibril formation of fish scale collagen facilitates early osteoblastic differentiation of human mesenchymal stem cells

J. Biomed. Mater Res. A

Skin, bone and muscle collagen extraction from the trash fish, leather jacket (Odonus niger) and their characterization

J. Food Sci. Technol.

Isolation of collagen from fish waste material —skin, bone, and fins

Food Chem.

Teleost fish scales: a unique biological model for the fabrication of materials for corneal stroma regeneration

J. Nanosci. Nanotechnol.

Cited by (30)

In vivo assessment of marine vs bovine origin collagen-based composite scaffolds promoting bone regeneration in a New Zealand rabbit model

Novel therapy using a fish scale collagen scaffold for rotator cuff healing in rat models

Fibril matrices created with collagen from the marine fish barramundi for use in conventional three-dimensional cell culture

Injectable chitosan/collagen hydrogels nano-engineered with functionalized single wall carbon nanotubes for minimally invasive applications in bone

Prionace glauca skin collagen bioengineered constructs as a promising approach to trigger cartilage regeneration

Simultaneous fabrication of carbon nanodots and hydroxyapatite nanoparticles from fish scale for biomedical applications