Effects of self-assembled type II collagen fibrils on the morphology and growth of pre-chondrogenic ATDC5 cells.

Objective: Although type II collagen could have marked potential for developing cartilage tissue engineering (CTE) scaffolds, its erratic supply and viscous nature have limited these studies, and there are no studies on the use of marine-derived type II collagen fibrils for CTE scaffold materials. In this study, we aimed to generate a fibril-based, thin-layered scaffold from marine-derived type II collagen and investigate its chondrogenic potential. Methods: Time-lapse observations revealed the cell adhesion process. The Cell Counting Kit-8 (CCK-8) assay, light microscopy, and scanning electron microscopy were performed to detect proliferation and filopodium morphology. Alcian blue staining was used to show the deposition of extracellular secretions, and qRT-PCR was performed to reveal the expression levels of chondrogenesis-related genes. Results: The cell adhesion speed was similar in both fibril-coated and control molecule-coated groups, but the cellular morphology, proliferation, and chondrogenesis activity differed. On fibrils, more elongated finer filopodia showed inter-cell communications, whereas the slower proliferation suggested an altered cell cycle. Extracellular secretions occurred before day 14 and continued until day 28 on fibrils, and on fibrils, the expression of the chondrogenesis-related genes Sox9 (p â€‹< â€‹0.001), Col10a1 (p â€‹< â€‹0.001), Acan (p â€‹< â€‹0.001), and Col2a1 (p â€‹= â€‹0.0049) was significantly upregulated on day 21. Conclusion: Marine-derived type II collagen was, for the first time, fabricated into a fibril state. It showed rapid cellular affinity and induced chondrogenesis with extracellular secretions. We presented a new model for studying chondrogenesis in vitro and a potential alternative material for cell-laden CTE research.

Industrial application of fish cartilaginous tissues.

Cartilage is primarily composed of proteoglycans and collagen. Bioactive compounds derived from animal cartilage, such as chondroitin sulfate and type II collagen, have multiple bioactivities and are incorporated in popular health products. The aging population and increases in degenerative and chronic diseases will stimulate the rapid growth of market demand for cartilage products. Commercial production of bioactive compounds primarily involves the cartilages of mammals and poultry. However, these traditional sources are associated zoonosis concerns; thus, cartilage products from the by-products of fish processing has gained increasing attention because of their high level of safety and other activities. In this review, we summarize the current state of research into fish-derived cartilage products and their application, and discuss future trends and tasks to encourage further expansion and exploitation. At present, shark cartilage is the primary source of marine cartilage. However, the number of shark catches is decreasing worldwide, owing to overfishing. This review considers the potential alternative fish cartilage sources for industrialization. Three keys, the sustainable production of fish, new fish-processing model, and market demand, have been discussed for the future realization of efficient fish cartilage use. The industrialization of fish-derived cartilage products is beneficial for achieving sustainable development of local economies and society.

Effects of phosphate ion concentration on in-vitro fibrillogenesis of sturgeon type I collagen.

Nonmammalian collagens have attracted significant attention owing to their potential for use as a source of cell scaffolds for tissue engineering. Since the morphology of collagen fibrils controls cell proliferation and differentiation, its regulation is essential for fabricating scaffolds with desirable characteristics. In this study, we evaluated the effects of the phosphate ion (Pi) concentration on the characteristics of fibrils formed from swim bladder type I collagen (SBC) and skin type I collagen (SC) from the Bester sturgeon. An increase in the Pi concentration decreased the fibril formation rate, promoted the formation of thick fibrils, and increased the thermal stability of the fibrils for both SBC and SC. However, the SBC and SC fibrils exhibited different fibril formation rates, degrees of fibrillogenesis, morphologies, and denaturation temperatures for the same reaction conditions. Finally, by regulating the Pi concentration, various types of SBC and SC fibrils could be coated on cell culture wells, and fibroblasts could be cultured on them. The results showed that thin fibrils enhance fibroblast extension and proliferation, whereas thick fibrils restrain fibroblast extension but orient them in the same direction. The results of this study suggest that SBC fibrils, which exhibit diverse morphologies, are suitable for use as a novel scaffold material, whose characteristics can be tailored readily by varying the Pi concentration.

Self-assembled collagen fibrils from the swim bladder of Bester sturgeon enable alignment of MC3T3-E1 cells and enhance osteogenic differentiation.

Collagen is the most abundant protein in animals, and its polymer, collagen fibrils, regulate cellular proliferation, differentiation, and migration. Low antigenicity, biocompatibility, and biodegradability make collagen fibrils suitable functional scaffolds for tissue engineering. In a previous study, we found that the type I atelocollagen purified from the swim bladder of Bester sturgeon (swim bladder collagen, SBC) showed high fibril-forming ability, producing thicker fibrils faster than porcine collagen. In this study, we report a novel method to coat cell culture wells with highly aligned collagen fibrils using the SBC. Two types of fibrils with different thicknesses were prepared by changing the crosslinking treatment timing. The oriented, thick collagen fibrils induced pre-osteoblastic MC3T3-E1, pre-adipocytic 3T3-L1, pre-myocytic C2C12, and fibroblastic L929 cells to align in the same direction, whereas the oriented, fine fibrils made a cell network with their long pseudopods. Cellular proliferation was inhibited on both fibrils. Furthermore, both fibrils induced the early differentiation of MC3T3-E1 cells without differentiation stimuli. In contrast, the morphology of pre-chondrocytic ATDC5 cells on both fine and thick fibrils extended very short pseudopods and continued to maintain a spherical shape without stretching, suggesting a distinct effect by the fibrils. The newly developed fibril coatings are in the form of a thin film, thereby providing good visibility of the cell structure, including cell-cell and cell-ECM interactions, using a phase contrast microscope. The fibril coatings have high potential as a useful tool for tissue engineering research.

Properties of collagen extracted from Amur sturgeon Acipenser schrenckii and assessment of collagen fibrils in vitro.

The objective of this study was to assess the nature of the collagens from the Amur sturgeon to determine its possibility as a potential collagen source for biomedical applications. From a sturgeon (1.22 kg), 6.0 g (dry wt) of skin collagen (SC), 4.1 g of swim bladder collagen (SBC), and 0.4 g of notochord collagen (NC) were obtained. SC and SBC were characterized as type I, and NC as type II collagen. Denaturation temperatures of SC, SBC, and NC were calculated as 28.5, 30.5, and 33.5 °C, respectively. Gene expression of the type I procollagen α2 chain of Amur sturgeon (ascol1a2) was specifically higher than ascol1a1 expression in the swim bladder, suggesting a unique composition of α chains in this organ. SC and SBC had better abilities of fibril formation with unique higher-order structures compared with porcine type I collagen. The maximum transition temperature (Tm) of reassembled fibrils formed in a buffer solution containing NaCl at 0 and 140 mM was 34.4 °C and 38.9 °C in SC, and 40.1 °C and 40.7 °C in SBC, respectively. These characteristic features suggested that sturgeon collagens could be used in the biomedical industries in future applications.

Antioxidant activity of proteins extracted from red alga dulse harvested in Japan.

In this study, we investigated antioxidant activity of proteins from the red alga dulse (Palmaria sp.) harvested in Hokkaido, Japan. The dulse proteins that contain phycoerythrin (PE) as the main component showed a high radical scavenging activity. To clarify the key constituent of antioxidant activity in dulse proteins, we prepared recombinant dulse PE ß-subunit (rPEß) (apoprotein) and chromophores from the dulse proteins. As a result, the rPEß showed lower radical scavenging activity than that of dulse proteins. On the other hand, the dulse chromophores composed mainly of phycoerythrobilin (PEB) indicated extremely higher radical scavenging activity (90.4% ± 0.1%) than that of dulse proteins (17.9% ± 0.1%) on ABTS assay. In addition, on cell viability assay using human neuroblastoma SH-SY5Y cells, the dulse chromophores showed extracellular and intracellular cytoprotective effects against H2 O2 -induced cell damage. From these data, we concluded that the dulse proteins have antioxidant ability and the activity principally derives from the chromophores. PRACTICAL APPLICATION: Dulse is an abundant and underused resource, which contains a lot of proteins, especially phycoerythrin. We here demonstrated that the practically prepared dulse proteins possessed antioxidant activity and clarified that chromophores from the dulse proteins were the key components. Therefore, the dulse proteins have a potential for functional material.

Anti-obesity effects of chondroitin sulfate oligosaccharides from the skate Raja pulchra.

We aimed to investigate the anti-obesity effects of chondroitin sulfate (CS) oligosaccharides obtained from cartilage of the skate Raja pulchra and to compare them with those of CSs of other molecular weights (MWts) (skate CS polysaccharides) and origins (shark CS, bovine CS). CSs suppressed pancreatic lipase activity as well as proliferation and lipid accumulation in mature adipocytes. Higher MWt CS had a greater lipase inhibitory activity than lower MWt CS. CSs of different origin show differing potencies for lipase inhibition and effects on adipocytes. Also, dietary intake of skate CS oligosaccharides could ameliorate obesity in high-fat diet mice model: it prevented gaining in body weight, liver weight and adipose tissue weight, maintained lower food consumption, inhibited intestinal absorption of triglyceride, and adjusted the serum endotoxin level. In conclusion, skate CS oligosaccharides have an anti-obesity activity, and the MWt and origin of the CSs may affect this activity.

The effect of alkaline pretreatment on the biochemical characteristics and fibril-forming abilities of types I and II collagen extracted from bester sturgeon by-products.

Non-mammalian collagens have attracted increasing attention for industrial and biomedical use. We have therefore evaluated extraction conditions and the biochemical properties of collagens from aquacultured sturgeon. Pepsin-soluble type I and type II collagen were respectively extracted from the skin and notochord of bester sturgeon by-products, with yields of 63.9 ±â€¯0.19% and 35.5 ±â€¯0.68%. Collagen extraction efficiency was improved by an alkaline pretreatment of the skin and notochord (fewer extraction cycles were required), but the final yields decreased to 56.2 ±â€¯0.84% for type I and 31.8 ±â€¯1.13% for type II. Alkaline pretreatment did not affect the thermal stability or triple-helical structure of both types of collagen. Types I and II collagen formed re-assembled fibril structures in vitro, under different conditions. Alkaline pretreatment slowed down the formation of type I collagen fibrils and specifically inhibited the formation of thick fibril-bundle structures. In contrast, alkaline pretreatment did not change type II collagen fibril formation. In conclusion, alkaline pretreatment of sturgeon skin and notochord is an effective method to accelerate collagen extraction process of types I and II collagen without changing their biochemical properties. However, it decreases the yield of both collagens and specifically changes the fibril-forming ability of type I collagen.

Detection and identification of furan fatty acids from fish lipids by high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry.

Using high-performance liquid chromatography coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS), we have developed a new method for detection and identification of furan fatty acids (F-acids), which are widely distributed in living organisms and foods as minor lipid components and are known to have antioxidant and anti-inflammatory effects. For this purpose, total fatty acids prepared from the testis lipids of Japanese chum salmon (Oncorhynchus keta) were examined without any concentration or isolation of F-acids. In negative ESI mode, F-acids gave a prominent [M-H]- ion, by which individual F-acids could be detected and identified. High-resolution extracted ion chromatograms clearly showed the occurrence of five major F-acid homologs as already reported by GC/MS. The method was successfully applied to several fish samples and revealed the occurrence of F-acids for the first time in the two New Zealand fish, hoki (Macruronus novaezelandiae) and school shark (Galeorhinus galeus).

Characterization of type I and II procollagen α1 chain in Amur sturgeon (Acipenser schrenckii) and comparison of their gene expression.

To characterize type I and II collagen in the Amur sturgeon at the molecular level, mRNAs encoding the proα chain of both types of collagen were cloned and sequenced. Full sequences of both were obtained, and the molecular phylogeny based on the deduced amino acid sequence indicated that the correct sequences of the target genes were obtained. Analyses of primary structure of the proα chains revealed that type I and II collagen share the basic structure of the proα chain of fibril collagen, but have different characteristics, especially in residues related to thermal stability. In the triple helical domain, Gly-Pro-Pro sequence stabilizing the tripeptide unit was more frequent in type II than in type I, and Gly-Gly, which likely decline in thermal stability, was more frequent in type I than in type II. These results suggested that the denaturation temperature of type II would be remarkably higher than type I. The spatial pattern of gene expression was analyzed by quantitative real-time PCR, which showed that relatively ubiquitous type I gene and strongly skewed distribution of type II gene, which highly expressed only in vertebra, snout cartilage, and notochord. This pattern was similar to the distribution pattern of each collagen protein detected by previous biochemical analyses using Amur and Bester sturgeons. The present study is the first report of the cloning of the full-length cDNAs for both of type I and type II collagen in the Amur sturgeon, and is the first comparative analysis of type I and II collagens in a sturgeon species at the molecular level. The results provide basic and general information on collagens in sturgeons.

Lack of hormonal stimulation prevents the landlocked Biwa salmon (Oncorhynchus masou subspecies) from adapting to seawater.

Landlocking of salmon relaxes selective pressures on hypoosmoregulatory ability (seawater adaptability) and may lead to the abandonment of its physiological system. However, little is known about the mechanism and consequence of the process. Biwa salmon is a strain/subspecies of Oncorhynchus masou that has been landlocked in Lake Biwa for an exceptionally long period (about 500,000 years) and has low ability to adapt to seawater. We compared activity of gill Na(+),K(+)-ATPase (NKA) of Biwa salmon with those of anadromous strains of the same species (masu and amago salmon) during downstream migration periods and after exogenous hormone treatment. Gill NKA activity in anadromous strains increased during their migration periods, while that in Biwa salmon remained low. However, treatments of Biwa salmon with growth hormone (GH) and cortisol increased gill NKA activity. Cortisol treatment also improved the whole body seawater adaptability of Biwa salmon. Receptors for GH and cortisol responded to hormonal treatments, whereas their mRNA levels during downstream migration period were essentially unchanged in Biwa salmon. Circulating levels of cortisol in masu salmon showed a peak during downstream migration period, while no such increase was seen in Biwa salmon. The present results indicate that Biwa salmon can improve its seawater adaptability by exogenous hormonal treatment, and hormone receptors are capable of responding to the signals. However, secretion of the endogenous hormone (cortisol) was not activated during the downstream migration period, which explains, at least in part, their low ability to adapt to seawater.

Biochemical characterisation and assessment of fibril-forming ability of collagens extracted from Bester sturgeon Huso huso × Acipenser ruthenus.

Collagens purified from Bester sturgeon organs were characterised biochemically, and their fibril-forming abilities and fibril morphologies formed in vitro clarified. Yields of collagens were 2.1%, 11.9%, 0.4%, 18.1%, 0.4%, 0.8% and 0.03% (collagen dry weight/tissue wet weight) from scales, skin, muscle, swim bladder, digestive tract, notochord and snout cartilage, respectively. Using SDS-PAGE and amino acid composition analyses, collagens from scales, skin, muscle, the swim bladder and digestive tract were characterised as type I, and collagens from the notochord and snout cartilage as type II. Denaturation temperatures of the collagens, measured using circular dichroism, were 29.6, 26.8, 29.0, 32.9, 31.6 and 36.3 °C in scales, skin, muscle, swim bladder, digestive tract, and notochord, respectively. For fibril formation, swim bladder and skin collagen showed a more rapid rate of increase in turbidity, a shorter time to attain the maximum turbidity, and formed thicker fibrils compared with porcine tendon type I collagen.

Spatiotemporal expression of the dermatopontin gene in zebrafish Danio rerio.

There has been significant interest in the expression and function of dermatopontin (DPT) in mammals owing to recent evidence pointing to its critical role in collagen fibrillogenesis. Despite this interest, limited information is available about the site/s of DPT mRNA expression or changes in expression in vivo. We used reverse-transcription polymerase chain reaction and in situ hybridization to evaluate the spatial and temporal pattern of DPT mRNA expression in zebrafish, Danio rerio, a widely used vertebrate model. We observed that DPT transcripts were expressed in zebrafish embryos at all developmental stages in a range of tissues, including the brain and optic neuron cells. Based on our results, we hypothesize that DPT may also play a role in neural functions in vivo.

Expression patterns of runx2, sparc, and bgp during scale regeneration in the goldfish Carassius auratus.

Teleost fish scale is a dermal skeleton equipped with a strong regenerative ability. Owing to this regenerative ability, teleost fish scale can be used as a model for the regeneration of the dermal skeleton. However, there is insufficient fundamental knowledge of the regeneration, and this limits the usage of fish scale. In this study, as a first step toward understanding the molecular mechanism of the cellular differentiation during scale regeneration, we cloned the cDNAs for osteoblast-related proteins (Runx2, Sparc, and Bgp) in goldfish, and analyzed their expressions during scale regeneration. The expression profiles of these genes during scale regeneration were similar to those during mammalian osteoblastic differentiation. Specifically, runx2 expression was increased at the earliest time point, followed by sparc expression and then bgp expression. In the earlier stages, these genes were expressed in cells that formed cellular condensations and the flat cells surrounding them in the scale pocket. As the regeneration proceeded, the expressions became restricted to the episquamal, hyposquamal, and marginal scleroblasts and the cells around the marginal area of the regenerating scale. These results strongly suggest that (1) the differentiation mechanism of scleroblasts is similar to that of mammalian osteoblasts and odontoblasts, (2) scleroblast differentiation occurs around the cellular condensations at the early regeneration stage and is restricted to the marginal area of the scale at the later stage, and (3) the differentiation mechanisms are similar between the episquamal scleroblasts that produce the external layer and the hyposquamal scleroblasts that produce the basal plate.

Immunohistochemical analysis of the proteolytic enzyme subtilase in the digestive organs of the sea urchin Strongylocentrotus intermedius.

Subtilase, a major protease in the short-spined sea urchin (Strongylocentrotus intermedius), was isolated and used as antigen for the subsequent production of a specific polyclonal antibody. Immunoreactive cells were observed by immunohistochemical analysis in granules in the anterior and posterior stomach and the anterior intestine. These granules, which were most numerous in the anterior stomach, also stained intensely with methylene blue-Azure II. However, granules in cells of the esophagus, posterior intestine, and rectum were not stained by this antibody. We conclude that subtilase mainly localizes in the stomach and anterior intestine of the sea urchin.

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

The corneal stroma is composed of multiple lamellae, each containing closely packed collagen fibrils. The orientation of fibrils in a lamella is parallel, but those in different lamellae are orthogonal. As a result, the corneal stroma has a characteristic orthogonal plywood-like structure. Such a highly-regulated three-dimensional arrangement of collagen fibrils gives strength and transparency to the corneal stroma, but it also presents a challenge in the fabrication of materials to replace it. A bioinspired technology is required to process such materials, but the regulatory mechanism of collagen-fibril orientation is still unknown. The low regenerating activity of the corneal stroma seems to be a major factor preventing progress in this field of research. A similarly highly-ordered arrangement of collagen fibrils can be seen in the basal plates of teleost fish scales. Moreover, the scales have high regenerating ability. When a scale is mechanically lost, a new scale is rapidly regenerated. The cells that produce the basal plates are extremely activated; thus, production of the highly-ordered collagen fibrils is very rapid. Therefore, the regenerating scales should be a uniquely helpful biological model for studying the regulatory mechanism of collagen-fibril orientation. Fish-scale collagen has another advantage for use as a biomaterial: the low probability of zoonotic infection. Therefore, scale collagen is a most promising biomaterial for fabricating three-dimensionally arranged collagen fibers to substitute for the corneal stroma. Three tasks that must be clarified for the bioinspired production of a corneal substitute from fish scale collagen are proposed.

A novel seminal plasma glycoprotein of a teleost, the Nile tilapia (Oreochromis niloticus), contains a partial von Willebrand factor type D domain and a zona pellucida-like domain.

Our previous study shows that seminal plasma of a teleost, the Nile tilapia, contains a glycoprotein Mr = 120,000 named as SPP (Seminal plasma glycoprotein)120 which forms a homopolymer that has sperm immobilizing activity. In order to elucidate the mechanisms of the formation of the homopolymer and the immobilization of sperm, molecular cloning of SPP120 was conducted. The cDNA for SPP120 contains a complete open reading frame encoding 797 amino acid residues with 14 potential N-glycosylation sites. The predicted amino acid sequence of SPP120 contains a partial von Willebrand factor type D domain and a zona pellucida domain, that are involved in protein-protein adhesion that form filamentous structures in various kinds of cells. This result suggests that SPP120 forms a homopolymer via these domains in seminal plasma and probably interacts with spermatozoa. Northern blotting reveals that the gene is also expressed in ovary, even in ovulated eggs. The results of in situ hybridization indicate that in testis the gene is expressed in Sertoli cells and epithelial cells of sperm ducts, and the localization corresponds to that of the protein analyzed by immunohistochemistry. In the ovary, the gene is expressed at the perinucleolus stage of oocytes; however, the protein is not detected in any cells other than oocytes.