Shizuka Yamada

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.

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.

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 μm. 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.

Chitosan and fish collagen as biomaterials for regenerative medicine.

This chapter focuses and reviews on the characteristics and biomedical application of chitosan and collagen from marine products and advantages and disadvantages of regeneration medicine. The understanding of the production processes of chitosan and collagen and the conformation of these biomaterials are indispensable for promoting the theoretical and practical availability. The initial inflammatory reactions associated with chitosan application to hard and soft tissues need to be controlled before it can be considered for clinical application as scaffold. Further, as chitosan takes too long for biodegradation in vivo, generally it is not suitable for the scaffold for degenerative medicine in especially dental pulp tissue. The collagen extract from the scales of tropical fish has been reported to have a degeneration temperature of 35°C. The properties of biocompatibility and biodegradation of fish atelocollagen are suitable for the scaffold in regenerative medicine.

Comparison of Conventional and New-generation Nickel-Titanium Files in Regard to Their Physical Properties

INTRODUCTION This study investigated the surface, fractured structure, and physicochemical properties related to cyclic fatigue in various nickel-titanium (NiTi) files. METHODS Among a total of 10 groups of NiTi files, conventional NiTi files (ProFile [Dentsply Maillefer, Ballaigues, Switzerland] and K3 [SybronEndo, Orange, CA]) and new-generation NiTi files (ProFile Vortex [PV; Tulsa Dental Specialties, Tulsa, OK], Vortex Blue [VB; Tulsa Dental Specialties], and K3 XF [XF; SybronEndo, Orange, CA]) with the same tip diameter (ISO size 25) and 2 types of taper (0.04 and 0.06) were used in this study. Scanning electron microscopy of the file surface structure, differential scanning calorimetry, and cyclic fatigue resistance tests were conducted. RESULTS Many mechanical grooves were recognized on the file surface. The surface in the ProFile group was extremely smooth compared with that observed for the other files. Many shallow hollows besides mechanical grooves were noted on the surface in the XF group. A smooth curve was observed in the ProFile, K3, and PV groups. Defined peaks in differential scanning calorimetry were observed in the VB and XF groups. The 0.04 taper files exhibited a statistically higher number of cycles to fracture than the 0.06 taper files in all groups (P < .05). Cracks along the mechanical grooves were observed in the NiTi files, with the exception of the XF group. The start of cracking was detected at U-shape sites in the ProFile group, the cutting edge in the PV and VB groups, and radial islands in the K3 and XF groups. CONCLUSIONS The present findings suggest that new-generation NiTi files are not necessarily improved compared with conventional files.

Zinc as an essential trace element in the acceleration of matrix vesicles-mediated mineral deposition

BACKGROUND: Zinc (Zn) has a potent stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. PURPOSE: The effect of Zn on the function of matrix vesicles (MVs) remains controversial. The purpose of this study was to investigate the effect of Zn on alkaline phosphatase (ALP) activity of osteoblasts and in the initial biological MVs‐mediated mineral deposition. STUDY DESIGN: Osteoblasts were treated with varying concentrations of Zn dissolved in culture medium. After three, five, and seven days of culture, ALP activity was assayed. For the detection of a low level of calcium concentration in MVs, X‐ray fluorescence (XRF) analyses were applied. The effect of Zn for the transformation of calcium phosphate was analyzed using a scanning electron microscope fitted with an energy dispersive X‐ray microanalysis (EDX) system. RESULTS: The ALP activity of osteoblasts in culture medium supplemented with 1 × 10−5M of Zn was significantly increased at both five and seven days. XRF data demonstrated higher levels of calcium concentration over time in the Zn‐supplemented group. EDX data showed that mineral deposits beginning on day 3 were transformed from whitlockite to calcium phosphate near hydroxyapatite, and that Zn accelerated this transformation. CONCLUSIONS: The proper concentration of Zn increased the ALP activity of osteoblasts after five and seven days of incubation. The present XRF and EDX data suggest that the increase of mineral deposition with Zn exposure for one to five days might be mediated by the activation of ALP and calcium‐binding proteins. Microsc. Res. Tech., 2011.

Effects of hypoxia on pluripotency in murine iPS cells

Retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c‐Myc) or three factors, excluding c‐Myc, has been shown to initiate a reprogramming process that results in the transformation of murine fibroblasts to induced pluripotent stem (iPS) cells, and there has been a rapid increase in the number of iPS cell‐based preclinical trials. In this study, the effects of these transcription factors were evaluated regarding the growth and differentiation of murine iPS cells under hypoxia. Based on the results of RT‐PCR and alizarin red S staining, there were no statistical differences in the growth and differentiation of iPS cells or the induction of iPS cells to osteoblasts under hypoxia between the transcription factor groups. Furthermore, the function of hypoxia inducible factors (HIFs) in murine iPS cells under hypoxia was investigated in relation to the morphology and expression of transcription factors using RT‐PCR and Western blotting. The HIF‐2α knockdown group exhibited a decrease in the colony size of the iPS cells. The HIF‐2α or ‐3α knockdown group demonstrated a statistically significant decrease in the transcription factor expression compared to that observed in the control group. These results demonstrate that HIF‐2α among HIFs is the most influential candidate for the maintenance of the pluripotency of murine iPS cells. Microsc. Res. Tech., 76:1084–1092, 2013.

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 (), 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 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 of TAC was 35-36°C. The scanning electron microscopy results indicated that the pore size (90–160 μm) 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.

Alfacalcidol enhances collagen quality in ovariectomized rat bones

The aim of this study was to investigate the effects of alfacalcidol (1α(OH)D3: ALF) on bone collagen employing an ovariectomized rat model. Thirty‐five 16‐week‐old female Sprague‐Dawley rats were divided into five groups: SHAM (sham‐operated + vehicle), OVX (ovariectomy + vehicle), and three ALF‐treated groups, that is, ovariectomy + 0.022 µg/kg/day ALF, ovariectomy + 0.067 µg/kg/day ALF, and ovariectomy + 0.2 µg/kg/day ALF. After 12 weeks of treatment, tibiae were subjected to histological, biochemical and immunohistochemical analyses. Collagen matrices in OVX bone appeared as immature and poorly organized; however, with the ALF treatment, it was improved in a dose‐dependent manner. Contents of collagen and pyridinoline cross‐link were decreased in OVX compared with SHAM, but they increased to the level comparable to SHAM in ALF‐treated groups. The total aldehyde, that is, a sum of free and those involved cross‐links, in the highest dose of ALF was significantly higher than the rest of the groups (p < 0.05). In addition, the expression of lysyl oxidase was increased in the all ALF‐treated groups compared with OVX (p < 0.05). In conclusion, ALF increases not only the amount of collagen but also enhances the maturation of collagen in ovariectomy‐induced osteoporotic bones, which likely contributes to the improvement of bone quality.

Early gene expression analyzed by a genome microarray and real-time PCR in osteoblasts cultured with a 4-META/MMA-TBB adhesive resin sealer

OBJECTIVES Adhesive resin sealer systems have been applied in endodontics to seal the root canal system. This study was designed to confirm the mechanism of intracellular molecular events in an in vitro cell culture system with a 4-methacryloxyethyl trimellitate anhydride/methylmethacrylate-tri-n-butyl borane (4-META/MMA-TBB) adhesive resin sealer. STUDY DESIGN The gene expression patterns relating to cell growth and differentiation were examined using a human genome expression microarray and real-time polymerase chain reaction analyses in hard tissue-forming osteoblasts cultured with and without a 4-META/MMA-TBB resin sealer. RESULTS There was no significant difference in the cell number between the control and adhesive sealer groups. An increased expression of integrin beta, transforming growth factor beta-related protein, craniofacial development protein 1, and PI3K genes was demonstrated. The integrin beta and PI3K genes showed extremely high ratios. CONCLUSIONS The signal transduction pathway, at least through the PI3K/Akt cascade for cell proliferation and differentiation, can be controlled by some components of this type of adhesive resin sealer.