Potency of Fish Collagen as a Scaffold for Regenerative Medicine

Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine

The Characterization of Fish (Tilapia) Collagen Sponge as a Biomaterial

For scaffold manufacturing, the utility of bioactive natural organic materials derived from marine products is useful and indispensable as an alternative to bovine collagen. The weakest feature of fish collagen for scaffold application is its low degeneration temperature (Td), indicating poor stability of fish collagen in mammals in vivo. We have focused on the tropical fish tilapia as a candidate for generating a clinical scaffold. The aim of this study was to confirm the Td of tilapia type I atelocollagen (TAC) for biomedical application. Furthermore, the physical and structural properties were investigated and evaluated as a scaffold on a sponge form. Different concentrations {0.5%, 1.0%, and 2.0% (v/v)} of TAC solution were analyzed. Differential scanning calorimetry showed that the Td of TAC was 35-36°C. The scanning electron microscopy results indicated that the pore size (90-160 m) of TAC sponges is acceptable for cell proliferation. The tensile strength of porous sponges was in the range of 0.01-0.07 MPa. These findings indicate that the TAC sponge prepared from tilapia is one of candidates as a scaffold. The 1.0% (v/v) concentration of TAC solution is especially recommended to be advantageous for preparing and handling the solution and for sponge formation

The E3 Ubiquitin Ligase Activity of Trip12 Is Essential for Mouse Embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development

D-Glucosamine Promotes Transfection Efficiency during Electroporation

D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures

Biological Safety of Fish (Tilapia) Collagen

Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine

Fabrication and Characteristics of Chitosan Sponge as a Tissue Engineering Scaffold

Cells, growth factors, and scaffolds are the three main factors required to create a tissue-engineered construct. After the appearance of bovine spongiform encephalopathy (BSE), considerable attention has therefore been focused on nonbovine materials. In this study, we examined the properties of a chitosan porous scaffold. A porous chitosan sponge was prepared by the controlled freezing and lyophilization of different concentrations of chitosan solutions. The materials were examined by scanning electron microscopy, and the porosity, tensile strength, and basic fibroblast growth factor (bFGF) release profiles from chitosan sponge were examined in vitro. The morphology of the chitosan scaffolds presented a typical microporous structure, with the pore size ranging from 50 to 200 m. The porosity of chitosan scaffolds with different concentrations was approximately 75-85%. A decreasing tendency for porosity was observed as the concentration of the chitosan increased. The relationship between the tensile properties and chitosan concentration indicated that the ultimate tensile strength for the sponge increased with a higher concentration. The in vitro bFGF release study showed that the higher the concentration of chitosan solution became, the longer the releasing time of the bFGF from the chitosan sponge was

Vapochromic Behaviour of M[Au(CN)2]2-Based Coordination Polymers (M = Co, Ni)

A series of M[Au(CN)2]2(analyte)x coordination polymers (M = Co, Ni; analyte = dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), pyridine; x = 2 or 4) was prepared and characterized. Addition of analyte vapours to solid M(μ-OH2)[Au(CN)2]2 yielded visible vapochromic responses for M = Co but not M = Ni; the IR νCN spectral region changed in every case. A single crystal structure of Zn[Au(CN)2]2(DMSO)2 revealed a corrugated 2-D layer structure with cis-DMSO units. Reacting a Ni(II) salt and K[Au(CN)2] in DMSO yielded the isostructural Ni[Au(CN)2]2(DMSO)2 product. Co[Au(CN)2]2(DMSO)2 and M[Au(CN)2]2(DMF)2 (M = Co, Ni) complexes have flat 2-D square-grid layer structures with trans-bound DMSO or DMF units; they are formed via vapour absorption by solid M(μ-OH2)[Au(CN)2]2 and from DMSO or DMF solution synthesis. Co[Au(CN)2]2(pyridine)4 is generated via vapour absorption by Co(μ-OH2)[Au(CN)2]2; the analogous Ni complex is synthesized by immersion of Ni(μ-OH2)[Au(CN)2]2 in 4% aqueous pyridine. Similar immersion of Co(μ-OH2)[Au(CN)2]2 yielded Co[Au(CN)2]2(pyridine)2, which has a flat 2-D square-grid structure with trans-pyridine units. Absorption of pyridine vapour by solid Ni(μ-OH2)[Au(CN)2]2 was incomplete, generating a mixture of pyridine-bound complexes. Analyte-free Co[Au(CN)2]2 was prepared by dehydration of Co(μ-OH2)[Au(CN)2]2 at 145 °C; it has a 3-D diamondoid-type structure and absorbs DMSO, DMF and pyridine to give the same materials as by vapour absorption from the hydrate

Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis.Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell.Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the transcriptional activity of AIB1

Effects of eluted components from 4-META/MMA-TBB adhesive resin sealer on osteoblastic cell proliferation

Background/purpose: Adhesive resin sealer systems are applied to seal root-canal systems more effectively through the formation of a resin impregnation layer. The purpose of this study was to investigate the effects of eluted components of the adhesive, Super-Bond sealer (SBS), on the proliferation of osteoblastic cells in vitro. Materials and methods: The standard powder:liquid ratio according to the manufacturer\u27s instructions was used to produce a cylindrical block of SBS (5 mm in diameter, 10 mm long) for this elution study. The resin block was placed on a 100-mm culture dish. Osteoblastic cells were seeded at a density of 4 × 10 6 in α-minimum essential medium containing 10% fetal bovine serum and cultured in a humidified incubator. After 3 days of culture with or without SBS, cells were retrieved and lysed according to the manufacturer\u27s instructions. The cellular events induced by the eluted components from SBS were analyzed using an antibody assay for mitogen-activated protein kinases (MAPKs) and a 3-(4,5-dimethylthyazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the concentration of boron, a component of the catalyst, tri-n-butyl borane (TBB), was analyzed using inductively coupled plasma optical emission spectrometry. Results: Expression of MAPKs increased after SBS application. The MTT assay indicated that TBB, one of the components of SBS, accelerated the proliferation of osteoblastic cells. Values of boron were 1.66 ± 0.37 and 1.74 ± 0.30 ppm in cells cultured with and without FBS, respectively. Conclusions: The eluted components from SBS can increase the expression of some MAPKs related to osteoblastic cell proliferation and differentiation in vitro. Both the elution experiment and treatment of cell culture with SBS components indicated that the boron originating from TBB is likely to be responsible for activation of the proliferation of osteoblastic cells